LOCUS       sequence-183245-        7088 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 7088)
  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 7088)
  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: MSP824
FEATURES             Location/Qualifiers
     source          1..7088
                     /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="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSMLIGGHKVFFVKPE
                     EPFQPVKEATQLMN"
     CDS             complement(869..886)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(923..2023)
                     /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(926..949)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      1057..1066
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             2029..2051
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2066..2090
                     /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(2090..2109)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2091..2109)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2193..2212)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2418..2495
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2496..3578
                     /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(2515..2534)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4387..4578
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4572..4594)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4680..4819
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4735..4754
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4834..4851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5005..5593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5085..5104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5704..5723)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5715..6530
                     /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             5715..6530
                     /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(5788..5807)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6622..7077)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6759..6780)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6971..6990
                     /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 tcacaaagaa aactttatgg cccccaatca
      241 acattgagtc 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 ttgtgtgccc atcagattca ttcgtcaaat gacaaatggt gctcgatatc gggttgtaat
      841 cacgcggccc cttaaacaag ctttctccat gatgatgatg atgatgactt tgcccataaa
      901 gattttctcc cgagctcgaa ttagtctgcg cgtctttcag ggcttcatcg acagtctgac
      961 gaccgctggc ggcgttgatc accgcagtac gcacggcata ccagaaagcg gacatctgcg
     1021 ggatgttcgg catgatttca cctttctggg cgttttccat ggtggcggca atacgtggat
     1081 ctttcgccaa ctcttcctcg taagacttca gcgctacggc acccagcggt ttgtctttat
     1141 taaccgcttc cagaccttca tcagtcagca gatagttttc gaggaactct tttgccagct
     1201 ctttgttcgg actggcggcg ttaatacctg cgctcagcac gccaacgaac ggtttggatg
     1261 gttgaccctt gaaggtcggc agtaccgtta caccataatt cactttgctg gtgtcgatgt
     1321 tggaccatgc ccacgggccg ttgatggtca tcgctgtttc gcctttatta aaggcagctt
     1381 ctgcgatgga gtaatcggtg tctgcattca tgtgtttgtt tttaatcagg tcaaccagga
     1441 aggtcagacc cgctttcgcg ccagcgttat ccacgcccac gtctttaatg tcgtacttgc
     1501 cgttttcata cttgaacgca taacccccgt cagcagcaat cagcggccag gtgaagtacg
     1561 gttcttgcag gttgaacatc agcgcgctct tacctttcgc tttcagttct ttatccagcg
     1621 ccgggatctc ttcccaggtt tttggcgggt tcggcagcag atctttgtta taaatcagcg
     1681 ataacgcttc aacagcgatc gggtaagcaa tcagcttgcc gttgtaacgt acggcatccc
     1741 aggtaaacgg atacagcttg tcctggaacg ctttgtccgg ggtgatttca gccaacaggc
     1801 cagattgagc gtagccacca aagcggtcgt gtgcccagaa gataatgtca gggccatcgc
     1861 cagttgccgc aacctgtggg aatttctctt ccagtttatc cggatgctca acggtgactt
     1921 taattccggt atctttctcg aatttcttac cgacttcagc gagaccgtta tagcctttat
     1981 cgccgttaat ccagattacc agtttacctt cttcgatttt catgtatatc tccttcttaa
     2041 agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga
     2101 gtcgtattaa tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg
     2161 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
     2221 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg
     2281 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
     2341 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     2401 agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     2461 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     2521 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     2581 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     2641 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     2701 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     2761 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     2821 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     2881 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     2941 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     3001 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     3061 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     3121 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     3181 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     3241 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     3301 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     3361 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     3421 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     3481 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     3541 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     3601 tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc
     3661 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     3721 atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc
     3781 tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc
     3841 ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt
     3901 atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     3961 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     4021 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     4081 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg
     4141 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     4201 cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
     4261 ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc
     4321 atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag
     4381 gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag
     4441 acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt
     4501 gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat
     4561 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     4621 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     4681 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
     4741 cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag
     4801 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4861 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4921 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4981 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5041 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5101 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5161 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5221 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5281 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5341 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5401 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     5461 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5521 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5581 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5641 aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta
     5701 atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa
     5761 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     5821 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
     5881 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     5941 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     6001 tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt
     6061 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     6121 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     6181 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     6241 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     6301 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     6361 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     6421 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     6481 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc
     6541 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6601 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     6661 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     6721 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     6781 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     6841 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     6901 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     6961 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     7021 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     7081 attcgcca
//
