LOCUS       sequence-182912-        7091 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression Vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7091)
  AUTHORS   Packer MS, Rees HA, Liu DR
  TITLE     Phage-assisted continuous evolution of proteases with altered 
            substrate specificity.
  JOURNAL   Nat Commun. 2017 Oct 16;8(1):956. doi: 10.1038/s41467-017-01055-9.
  PUBMED    29038472
REFERENCE   2  (bases 1 to 7091)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2017-10-16"; volume: "8(1):956. doi"; pages: " 
            10.1038/s41467-017-01055-9"
COMMENT     Sequence Label: MSP968
FEATURES             Location/Qualifiers
     source          1..7091
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(161..868)
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGLTASLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     CDS             complement(869..889)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             complement(890..910)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             complement(926..2026)
                     /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."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(929..952)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      1060..1069
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             2032..2054
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2069..2093
                     /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)."
     primer_bind     complement(2093..2112)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2094..2112)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2196..2215)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2421..2498
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2499..3581
                     /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_bind     complement(2518..2537)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4390..4581
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4575..4597)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4683..4822
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4738..4757
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4837..4854)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5008..5596)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5088..5107)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5707..5726)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5718..6533
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5718..6533
                     /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"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5791..5810)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6625..7080)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6762..6783)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6974..6993
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgtta attcatgagt tgagtcgctt
      181 ccttaactgg ctgaaaaggc tcttcaggtt tcaccatgaa aactttatgg cccccccaca
      241 atactgagtc agcatttaat cgccaaccac taacccactg ctgcgcctcc tgatttgtca
      301 acaattccat gaagtttttc ggcacgcttg tgaaataatt gtttgtgttg gtgaaattcg
      361 atgctgagtg tataccaaca atgaacccat ctctagttga tactaatgga ctgccacact
      421 gcccatcctt ggtttgaatc caatgcttcc agaatatgcc atcagatgaa gggaatgtgc
      481 aactagtgtc tgacaccatg ctagacatgc tcttagtttg gaagttggtt gtcacaagac
      541 atatgcgctc ttccctttgt ggctctctaa atttcagctt ttgaggaaat ggtgggaaat
      601 ccttaggcat gcgaataatt atcatgtccc tcccatcaat gaggtgttgt tgcaaagtcg
      661 tggtgttctt gaccttgaat acaccatgta gtgattggac caacagtgtt ccattatttc
      721 ttctaaacaa gtgcttgttt gtaatgatga agggaccaaa tccaatacca tacaacgatg
      781 ctgtgagccc atcagattca ttcgtcaaat gacaaatggt gctcgatatc gggttgtaat
      841 cacgcggccc cttaaacaag ctttctccat gatgatgatg atgatgatgc atttgaaaat
      901 aaagattttc tcccgagctc gaattagtct gcgcgtcttt cagggcttca tcgacagtct
      961 gacgaccgct ggcggcgttg atcaccgcag tacgcacggc ataccagaaa gcggacatct
     1021 gcgggatgtt cggcatgatt tcacctttct gggcgttttc catggtggcg gcaatacgtg
     1081 gatctttcgc caactcttcc tcgtaagact tcagcgctac ggcacccagc ggtttgtctt
     1141 tattaaccgc ttccagacct tcatcagtca gcagatagtt ttcgaggaac tcttttgcca
     1201 gctctttgtt cggactggcg gcgttaatac ctgcgctcag cacgccaacg aacggtttgg
     1261 atggttgacc cttgaaggtc ggcagtaccg ttacaccata attcactttg ctggtgtcga
     1321 tgttggacca tgcccacggg ccgttgatgg tcatcgctgt ttcgccttta ttaaaggcag
     1381 cttctgcgat ggagtaatcg gtgtctgcat tcatgtgttt gtttttaatc aggtcaacca
     1441 ggaaggtcag acccgctttc gcgccagcgt tatccacgcc cacgtcttta atgtcgtact
     1501 tgccgttttc atacttgaac gcataacccc cgtcagcagc aatcagcggc caggtgaagt
     1561 acggttcttg caggttgaac atcagcgcgc tcttaccttt cgctttcagt tctttatcca
     1621 gcgccgggat ctcttcccag gtttttggcg ggttcggcag cagatctttg ttataaatca
     1681 gcgataacgc ttcaacagcg atcgggtaag caatcagctt gccgttgtaa cgtacggcat
     1741 cccaggtaaa cggatacagc ttgtcctgga acgctttgtc cggggtgatt tcagccaaca
     1801 ggccagattg agcgtagcca ccaaagcggt cgtgtgccca gaagataatg tcagggccat
     1861 cgccagttgc cgcaacctgt gggaatttct cttccagttt atccggatgc tcaacggtga
     1921 ctttaattcc ggtatctttc tcgaatttct taccgacttc agcgagaccg ttatagcctt
     1981 tatcgccgtt aatccagatt accagtttac cttcttcgat tttcatgtat atctccttct
     2041 taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag
     2101 tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc
     2161 cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg
     2221 ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc
     2281 aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc
     2341 ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa
     2401 gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
     2461 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     2521 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     2581 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     2641 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     2701 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     2761 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     2821 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     2881 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     2941 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     3001 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     3061 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     3121 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     3181 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     3241 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     3301 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     3361 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     3421 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     3481 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     3541 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
     3601 agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca
     3661 gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc
     3721 tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac
     3781 cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac
     3841 gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc
     3901 attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc
     3961 cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga
     4021 acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc
     4081 ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc
     4141 cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg
     4201 aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt
     4261 tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg
     4321 agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg
     4381 gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc
     4441 cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc
     4501 tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt
     4561 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     4621 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     4681 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
     4741 catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc acagatgcgt
     4801 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4861 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4921 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4981 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5041 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5101 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5161 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5221 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5281 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5341 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5401 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     5461 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5521 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5581 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5641 cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct tacataaaca
     5701 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat
     5761 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     5821 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     5881 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     5941 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     6001 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg
     6061 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     6121 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     6181 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     6241 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     6301 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     6361 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     6421 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     6481 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa
     6541 ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6601 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     6661 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6721 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     6781 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     6841 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     6901 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     6961 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     7021 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     7081 cccattcgcc a
//
