LOCUS       pET28a(+)-MBP-TE        6762 bp ds-DNA     circular SYN 15-APR-2021
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6762)
  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|946857600|
COMMENT     VNTDBDATE|946857600|
COMMENT     VNTNAME|pET28a(+)-MBP-TEV-Esolu1|
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..1828
                     /codon_start=1
                     /label=Esolu1
     CDS             1835..1852
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
     primer          complement(1905..1923)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
                     /note="sequence: GCTAGTTATTGCTCAGCGG"
     terminator      1919..1966
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2003..2458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer          complement(2090..2109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="sequence: AGGGAAGAAAGCGAAAGGAG"
     primer          2300..2321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="sequence: GTGGACTCTTGTTCCAAACTGG"
     CDS             complement(2550..3365)
                     /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          3273..3292
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
                     /note="sequence: ATCGCGAGCCCATTTATACC"
     primer          3357..3376
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
                     /note="sequence: ATGGCTCATAACACCCCTTG"
     rep_origin      3487..4075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer          3976..3995
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="sequence: GGGAAACGCCTGGTATCTTT"
     primer          4229..4246
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="sequence: AGCGAGTCAGTGAGCGAG"
     misc_feature    4261..4400
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer          complement(4326..4345)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /note="sequence: CGGCATCAGAGCAGATTGTA"
     primer          4486..4508
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
                     /note="sequence: CCGGGAGCTGCATGTGTCAGAGG"
     CDS             complement(4502..4693)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5502..6584)
                     /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          6546..6565
                     /label=LacI-R
                     /note="LacI, reverse primer"
                     /note="sequence: GGCATACTCTGCGACATCGT"
     promoter        complement(6585..6662)
                     /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 atccagcttt gcaggtctga aagatgcaga tgttgcagca gcactggcag catgtagcgc
     1561 agcagatagc tttaaacaca aagagttttt tgccaaagtt ggcctggcaa gcaaaagcct
     1621 ggatgatgtt aaaaaagcct tctatgtgat cgaccaggat aaaagcggtt tcatcgaaga
     1681 agatgaactg aaactgttcc tgcagaattt tagcccgagc gcacgtgcac tgaccgatgc
     1741 agaaaccaaa gcatttctgg cagatggtga taaagatggt gacggtatga ttggtgttga
     1801 tgaatttgca gcaatgatca aggcctaact cgagcaccac caccaccacc actgagatcc
     1861 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1921 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     1981 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2041 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2281 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2341 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2401 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2461 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2521 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     2581 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     2641 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     2701 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     2761 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     2821 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     2881 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     2941 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3001 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     3061 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3121 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     3181 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     3241 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3301 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     3361 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     3421 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3481 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3541 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3601 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3661 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3721 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3781 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3841 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3901 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3961 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4021 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4081 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4141 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4201 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4261 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     4321 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4381 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4441 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4501 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     4561 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     4621 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     4681 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     4741 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     4801 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4861 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4921 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4981 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     5041 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     5101 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     5161 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5221 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5281 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5341 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5401 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5461 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5521 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5581 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     5641 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     5701 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     5761 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     5821 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     5881 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     5941 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6001 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6061 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     6121 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6181 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6241 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6301 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6361 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6421 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6481 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6541 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     6601 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     6661 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     6721 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tg
//
