LOCUS       pET28a(+)-MBP-TE        6768 bp ds-DNA     circular SYN 15-APR-2021
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6768)
  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-Oncm1|
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..1834
                     /codon_start=1
                     /label=Oncm1
     CDS             1841..1858
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
     primer          complement(1911..1929)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
                     /note="sequence: GCTAGTTATTGCTCAGCGG"
     terminator      1925..1972
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2009..2464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer          complement(2096..2115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="sequence: AGGGAAGAAAGCGAAAGGAG"
     primer          2306..2327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="sequence: GTGGACTCTTGTTCCAAACTGG"
     CDS             complement(2556..3371)
                     /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          3279..3298
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
                     /note="sequence: ATCGCGAGCCCATTTATACC"
     primer          3363..3382
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
                     /note="sequence: ATGGCTCATAACACCCCTTG"
     rep_origin      3493..4081
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer          3982..4001
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="sequence: GGGAAACGCCTGGTATCTTT"
     primer          4235..4252
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="sequence: AGCGAGTCAGTGAGCGAG"
     misc_feature    4267..4406
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer          complement(4332..4351)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /note="sequence: CGGCATCAGAGCAGATTGTA"
     primer          4492..4514
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
                     /note="sequence: CCGGGAGCTGCATGTGTCAGAGG"
     CDS             complement(4508..4699)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5508..6590)
                     /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          6552..6571
                     /label=LacI-R
                     /note="LacI, reverse primer"
                     /note="sequence: GGCATACTCTGCGACATCGT"
     promoter        complement(6591..6668)
                     /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 atccatggca tgtgcccatc tgtgtaaaga agcagatatc aaaaccgcac tggaagcatg
     1561 taaagcagcc gatagcttta acttcaaaac cttttttcac accattggct ttgcaagcaa
     1621 aagcgcagat gatgtcaaaa aagccttcaa agtgattgat caggatgcca gcggttttat
     1681 tgaagtggaa gaactgaaac tgttcctgca gaatttttgt ccgaaagcac gtgttctgac
     1741 cgatgcagaa accaaagcat ttctgaaagc cggtgatgca gatggtgatg gtatgattgg
     1801 tattgatgaa tttgccgttt gggtgaagca gtaactcgag caccaccacc accaccactg
     1861 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1921 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1981 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     2041 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2101 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2161 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2221 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2281 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2341 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2401 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2461 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2521 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     2581 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2641 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2701 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2761 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     2821 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     2881 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     2941 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     3001 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     3061 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     3121 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     3181 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3241 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3301 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     3361 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3421 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3481 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3541 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3601 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3661 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3721 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3781 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3841 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3901 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3961 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4021 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4081 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4141 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4201 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4261 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     4321 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4381 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4441 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4501 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     4561 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     4621 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     4681 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     4741 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4801 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     4861 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     4921 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     4981 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5041 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5101 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5161 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     5221 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     5281 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     5341 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     5401 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     5461 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     5521 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5581 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     5641 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     5701 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     5761 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     5821 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     5881 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     5941 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     6001 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     6061 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     6121 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     6181 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     6241 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     6301 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     6361 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     6421 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     6481 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     6541 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     6601 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     6661 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     6721 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatg
//
