LOCUS       sequence-182914-        7090 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 7090)
  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 7090)
  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: MSP969
FEATURES             Location/Qualifiers
     source          1..7090
                     /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(868..888)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             complement(925..2025)
                     /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(928..951)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      1059..1068
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             2031..2053
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2068..2092
                     /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(2092..2111)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2093..2111)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2195..2214)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2420..2497
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2498..3580
                     /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(2517..2536)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4389..4580
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4574..4596)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4682..4821
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4737..4756
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4836..4853)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5007..5595)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5087..5106)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5706..5725)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5717..6532
                     /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             5717..6532
                     /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(5790..5809)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6624..7079)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6761..6782)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6973..6992
                     /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 cttaactggc tgaaaaggct cttcaggttt caccatgaaa actttatggc ccccccacaa
      241 tactgagtca gcatttaatc gccaaccact aacccactgc tgcgcctcct gatttgtcaa
      301 caattccatg aagtttttcg gcacgcttgt gaaatacatg tttgtgttgg tgaaattcgc
      361 tgctgagtgt ataccaacaa tgaacccatc tctagttgat actaatggat tgccacactg
      421 cccaggcttg gattgaatcc aatgcttcca gaatatgcca tcagatgaag ggaatgtgca
      481 actagtgtct gacaccatgc tagacatgct cttagtttgg aagttggttg tcacaagaca
      541 tatgcgctct tccctttgtg gctctctaaa tttcagcttt tgaggaaatg gtgggaaatc
      601 cttaggcatg cgaataatta tcatgtccct cccatcaatg aggtgttgtt gcaaagtcgt
      661 ggtgttcttg accttgaata caccatgtag tgattggacc aacagtgttc cattatttct
      721 tctaaacaag tgcttgtttg taatgatgaa gggaccaaat ccaataccat acaacgatgt
      781 tgtgtgccca tcagattcat tcgtcaaatg acaaatggtg ctcgatatcg ggttgtaatc
      841 acgcggcccc ttaaacaagc tttctccatg atgatgatga tgatgatgac tgtgaaaata
      901 aagattttct cccgagctcg aattagtctg cgcgtctttc agggcttcat cgacagtctg
      961 acgaccgctg gcggcgttga tcaccgcagt acgcacggca taccagaaag cggacatctg
     1021 cgggatgttc ggcatgattt cacctttctg ggcgttttcc atggtggcgg caatacgtgg
     1081 atctttcgcc aactcttcct cgtaagactt cagcgctacg gcacccagcg gtttgtcttt
     1141 attaaccgct tccagacctt catcagtcag cagatagttt tcgaggaact cttttgccag
     1201 ctctttgttc ggactggcgg cgttaatacc tgcgctcagc acgccaacga acggtttgga
     1261 tggttgaccc ttgaaggtcg gcagtaccgt tacaccataa ttcactttgc tggtgtcgat
     1321 gttggaccat gcccacgggc cgttgatggt catcgctgtt tcgcctttat taaaggcagc
     1381 ttctgcgatg gagtaatcgg tgtctgcatt catgtgtttg tttttaatca ggtcaaccag
     1441 gaaggtcaga cccgctttcg cgccagcgtt atccacgccc acgtctttaa tgtcgtactt
     1501 gccgttttca tacttgaacg cataaccccc gtcagcagca atcagcggcc aggtgaagta
     1561 cggttcttgc aggttgaaca tcagcgcgct cttacctttc gctttcagtt ctttatccag
     1621 cgccgggatc tcttcccagg tttttggcgg gttcggcagc agatctttgt tataaatcag
     1681 cgataacgct tcaacagcga tcgggtaagc aatcagcttg ccgttgtaac gtacggcatc
     1741 ccaggtaaac ggatacagct tgtcctggaa cgctttgtcc ggggtgattt cagccaacag
     1801 gccagattga gcgtagccac caaagcggtc gtgtgcccag aagataatgt cagggccatc
     1861 gccagttgcc gcaacctgtg ggaatttctc ttccagttta tccggatgct caacggtgac
     1921 tttaattccg gtatctttct cgaatttctt accgacttca gcgagaccgt tatagccttt
     1981 atcgccgtta atccagatta ccagtttacc ttcttcgatt ttcatgtata tctccttctt
     2041 aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt
     2101 gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc
     2161 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg
     2221 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca
     2281 ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg
     2341 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag
     2401 ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg
     2461 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga
     2521 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag
     2581 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     2641 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac
     2701 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     2761 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     2821 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     2881 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     2941 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     3001 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     3061 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa
     3121 tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca
     3181 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca
     3241 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat
     3301 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac
     3361 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc
     3421 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
     3481 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     3541 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa
     3601 gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag
     3661 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
     3721 ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc
     3781 gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg
     3841 ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca
     3901 ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc
     3961 ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa
     4021 cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg
     4081 gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc
     4141 ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga
     4201 agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt
     4261 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
     4321 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
     4381 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
     4441 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
     4501 gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
     4561 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     4621 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     4681 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     4741 atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta
     4801 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4861 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4921 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4981 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5041 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5101 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5161 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5221 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5281 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5341 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5401 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     5461 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5521 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5581 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5641 gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag
     5701 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt
     5761 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     5821 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
     5881 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     5941 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     6001 gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga
     6061 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     6121 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     6181 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     6241 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     6301 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     6361 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     6421 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     6481 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat
     6541 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6601 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     6661 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     6721 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     6781 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     6841 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     6901 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     6961 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     7021 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     7081 ccattcgcca
//
