LOCUS       sequence-182919-        7001 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 7001)
  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 7001)
  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: MSP574
FEATURES             Location/Qualifiers
     source          1..7001
                     /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(779..799)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             complement(800..820)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(836..1936)
                     /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(839..862)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      970..979
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             1942..1964
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1979..2003
                     /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(2003..2022)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2004..2022)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2106..2125)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2331..2408
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2409..3491
                     /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(2428..2447)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4300..4491
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4485..4507)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4593..4732
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4648..4667
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4747..4764)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4918..5506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4998..5017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5617..5636)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5628..6443
                     /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             5628..6443
                     /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(5701..5720)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6535..6990)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6672..6693)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6884..6903
                     /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 ggtggtgctg cttgccgttt ccgttaatcg
      181 ggcggtttac gtgttcaggt gacgctaaga aagctttgat cttcggacgg gcgctcaggc
      241 gcgctacata tgcgctcagt aacgggaaag cgtctaaaca gctaggattt aaaacctggt
      301 gaatgcgtaa taagtctaat aagttataat cagcgaagct aatttggcta ccaactacga
      361 acgcctgacc tccctgattc tggctcagta atgtttcaaa cggcttcagg tgttcaggta
      421 attctttcac gtacttctct ttaccggctt cataattagt gtaaattaac gtcgcgtatt
      481 tacaacgtaa gtcttccacg ccatcattca ccatatccac taaggctgct tctttctgat
      541 ctttaccgta taaaccaaat gagcgaccta aatgacgtaa gatcgcgtta gactggtata
      601 aggttaaatc accatcctga aacttcggta attgacggaa taaacagcta ggctttaacg
      661 gcggccaggt ttccattgtc actacttctt ccttccatga ttgatcctgg tccgctaata
      721 acatacgcat cgcttcacaa cgaccacgta ccggaaagta tgtaatggtg tacggcggat
      781 gatgatgatg atgatgatga ctttgaaaat aaagattttc tcccgagctc gaattagtct
      841 gcgcgtcttt cagggcttca tcgacagtct gacgaccgct ggcggcgttg atcaccgcag
      901 tacgcacggc ataccagaaa gcggacatct gcgggatgtt cggcatgatt tcacctttct
      961 gggcgttttc catggtggcg gcaatacgtg gatctttcgc caactcttcc tcgtaagact
     1021 tcagcgctac ggcacccagc ggtttgtctt tattaaccgc ttccagacct tcatcagtca
     1081 gcagatagtt ttcgaggaac tcttttgcca gctctttgtt cggactggcg gcgttaatac
     1141 ctgcgctcag cacgccaacg aacggtttgg atggttgacc cttgaaggtc ggcagtaccg
     1201 ttacaccata attcactttg ctggtgtcga tgttggacca tgcccacggg ccgttgatgg
     1261 tcatcgctgt ttcgccttta ttaaaggcag cttctgcgat ggagtaatcg gtgtctgcat
     1321 tcatgtgttt gtttttaatc aggtcaacca ggaaggtcag acccgctttc gcgccagcgt
     1381 tatccacgcc cacgtcttta atgtcgtact tgccgttttc atacttgaac gcataacccc
     1441 cgtcagcagc aatcagcggc caggtgaagt acggttcttg caggttgaac atcagcgcgc
     1501 tcttaccttt cgctttcagt tctttatcca gcgccgggat ctcttcccag gtttttggcg
     1561 ggttcggcag cagatctttg ttataaatca gcgataacgc ttcaacagcg atcgggtaag
     1621 caatcagctt gccgttgtaa cgtacggcat cccaggtaaa cggatacagc ttgtcctgga
     1681 acgctttgtc cggggtgatt tcagccaaca ggccagattg agcgtagcca ccaaagcggt
     1741 cgtgtgccca gaagataatg tcagggccat cgccagttgc cgcaacctgt gggaatttct
     1801 cttccagttt atccggatgc tcaacggtga ctttaattcc ggtatctttc tcgaatttct
     1861 taccgacttc agcgagaccg ttatagcctt tatcgccgtt aatccagatt accagtttac
     1921 cttcttcgat tttcatgtat atctccttct taaagttaaa caaaattatt tctagagggg
     1981 aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat
     2041 ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc
     2101 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat
     2161 gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc
     2221 catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact
     2281 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg
     2341 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     2401 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     2461 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     2521 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     2581 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     2641 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     2701 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     2761 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     2821 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     2881 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     2941 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     3001 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     3061 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     3121 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     3181 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     3241 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     3301 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     3361 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     3421 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     3481 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct
     3541 cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg
     3601 actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg
     3661 gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc
     3721 ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc
     3781 gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg
     3841 cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt
     3901 tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct
     3961 gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg
     4021 cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta
     4081 ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt
     4141 ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg
     4201 gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt
     4261 acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc
     4321 gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac
     4381 gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag
     4441 ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     4501 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     4561 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
     4621 agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4681 attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     4741 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4801 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4861 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4921 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4981 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5041 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5101 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5161 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5221 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5281 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5341 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5401 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5461 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5521 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5581 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     5641 aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg
     5701 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
     5761 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     5821 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     5881 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa
     5941 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     6001 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     6061 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     6121 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     6181 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     6241 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     6301 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     6361 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     6421 tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt
     6481 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     6541 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     6601 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     6661 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     6721 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     6781 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     6841 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     6901 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     6961 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc a
//
