LOCUS       pET28a(+)-MBP-TE        6792 bp ds-DNA     circular SYN 07-APR-2021
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6792)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jun 24, 2021 from SnapGene Viewer 5.1.7 to Vector
            NTI(R) format
            https://www.snapgene.com
COMMENT     VNTDATE|945216000|
COMMENT     VNTDBDATE|946166400|
COMMENT     VNTNAME|pET28a(+)-MBP-TEV-Blot5|
COMMENT     VNTAUTHORNAME|Trial User|
FEATURES             Location/Qualifiers
     primer          106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
                     /note="sequence: GGTGATGTCGGCGATATAGG"
     primer          209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
                     /note="sequence: TAATACGACTCACTATAGGG"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             267..289
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
     CDS             335..352
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
     CDS             368..1465
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
     regulatory      1322..1331
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer          1439..1462
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
                     /note="sequence: GATGAAGCCCTGAAAGACGCGCAG"
     CDS             1481..1499
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
     CDS             1505..1858
                     /codon_start=1
                     /label=Blot5
     CDS             1865..1882
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
     primer          complement(1935..1953)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
                     /note="sequence: GCTAGTTATTGCTCAGCGG"
     terminator      1949..1996
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2033..2488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer          complement(2120..2139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="sequence: AGGGAAGAAAGCGAAAGGAG"
     primer          2330..2351
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="sequence: GTGGACTCTTGTTCCAAACTGG"
     CDS             complement(2580..3395)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
     primer          3303..3322
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
                     /note="sequence: ATCGCGAGCCCATTTATACC"
     primer          3387..3406
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
                     /note="sequence: ATGGCTCATAACACCCCTTG"
     rep_origin      3517..4105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer          4006..4025
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="sequence: GGGAAACGCCTGGTATCTTT"
     primer          4259..4276
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="sequence: AGCGAGTCAGTGAGCGAG"
     misc_feature    4291..4430
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer          complement(4356..4375)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /note="sequence: CGGCATCAGAGCAGATTGTA"
     primer          4516..4538
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
                     /note="sequence: CCGGGAGCTGCATGTGTCAGAGG"
     CDS             complement(4532..4723)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5532..6614)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer          6576..6595
                     /label=LacI-R
                     /note="LacI, reverse primer"
                     /note="sequence: GGCATACTCTGCGACATCGT"
     promoter        complement(6615..6692)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatggc
      361 tagcggtaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
      421 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
      481 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
      541 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
      601 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
      661 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
      721 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
      781 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
      841 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
      901 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
      961 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
     1021 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
     1081 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
     1141 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcgaaaga
     1201 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     1261 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
     1321 cgccaccatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     1381 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
     1441 tgaagccctg aaagacgcgc agactcgtat caccaagggt gaaaatctat acttccaagg
     1501 atcccaagaa cacaaaccga aaaaagatga ttttcgcaac gagttcgatc atctgctgat
     1561 tgaacaggca aatcatgcca ttgaaaaagg tgaacatcag ctgctgtatc tgcagcatca
     1621 gctggatgaa ctgaatgaga ataaaagcaa agagctgcaa gaaaagatta tccgtgaact
     1681 ggatgttgtt tgcgcaatga ttgaaggtgc acagggtgca ctggaacgtg aactgaaacg
     1741 taccgatctg aatattctgg aacgcttcaa ttatgaagaa gcacagaccc tgagcaaaat
     1801 cctgctgaaa gatctgaaag aaaccgagca gaaagtgaaa gatattcaga cccagtaact
     1861 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     1921 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1981 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     2041 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2101 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2161 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2221 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2281 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2341 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2401 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2461 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     2521 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     2581 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     2641 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     2701 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     2761 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     2821 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     2881 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     2941 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     3001 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     3061 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     3121 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     3181 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     3241 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     3301 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     3361 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     3421 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3481 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3541 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3601 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3661 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3721 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3781 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3841 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3901 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3961 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4021 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4081 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4141 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4201 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4261 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     4321 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4381 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     4441 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4501 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4561 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     4621 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     4681 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4741 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4801 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4861 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4921 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4981 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     5041 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     5101 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     5161 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     5221 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     5281 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     5341 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     5401 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     5461 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     5521 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5581 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     5641 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5701 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5761 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     5821 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5881 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5941 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     6001 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     6061 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     6121 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     6181 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     6241 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     6301 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     6361 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     6421 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     6481 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     6541 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     6601 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6661 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     6721 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     6781 gaatggtgca tg
//
