LOCUS       sequence-183210-        6998 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 6998)
  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 6998)
  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: MSP809
FEATURES             Location/Qualifiers
     source          1..6998
                     /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..796)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(833..1933)
                     /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(836..859)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      967..976
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             1939..1961
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1976..2000
                     /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(2000..2019)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2001..2019)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2103..2122)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2328..2405
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2406..3488
                     /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(2425..2444)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4297..4488
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4482..4504)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4590..4729
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4645..4664
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4744..4761)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4915..5503)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4995..5014)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5614..5633)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5625..6440
                     /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             5625..6440
                     /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(5698..5717)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6532..6987)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6669..6690)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6881..6900
                     /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 atgatgcatg tgaccaacca gcgggtgtcc cgagctcgaa ttagtctgcg
      841 cgtctttcag ggcttcatcg acagtctgac gaccgctggc ggcgttgatc accgcagtac
      901 gcacggcata ccagaaagcg gacatctgcg ggatgttcgg catgatttca cctttctggg
      961 cgttttccat ggtggcggca atacgtggat ctttcgccaa ctcttcctcg taagacttca
     1021 gcgctacggc acccagcggt ttgtctttat taaccgcttc cagaccttca tcagtcagca
     1081 gatagttttc gaggaactct tttgccagct ctttgttcgg actggcggcg ttaatacctg
     1141 cgctcagcac gccaacgaac ggtttggatg gttgaccctt gaaggtcggc agtaccgtta
     1201 caccataatt cactttgctg gtgtcgatgt tggaccatgc ccacgggccg ttgatggtca
     1261 tcgctgtttc gcctttatta aaggcagctt ctgcgatgga gtaatcggtg tctgcattca
     1321 tgtgtttgtt tttaatcagg tcaaccagga aggtcagacc cgctttcgcg ccagcgttat
     1381 ccacgcccac gtctttaatg tcgtacttgc cgttttcata cttgaacgca taacccccgt
     1441 cagcagcaat cagcggccag gtgaagtacg gttcttgcag gttgaacatc agcgcgctct
     1501 tacctttcgc tttcagttct ttatccagcg ccgggatctc ttcccaggtt tttggcgggt
     1561 tcggcagcag atctttgtta taaatcagcg ataacgcttc aacagcgatc gggtaagcaa
     1621 tcagcttgcc gttgtaacgt acggcatccc aggtaaacgg atacagcttg tcctggaacg
     1681 ctttgtccgg ggtgatttca gccaacaggc cagattgagc gtagccacca aagcggtcgt
     1741 gtgcccagaa gataatgtca gggccatcgc cagttgccgc aacctgtggg aatttctctt
     1801 ccagtttatc cggatgctca acggtgactt taattccggt atctttctcg aatttcttac
     1861 cgacttcagc gagaccgtta tagcctttat cgccgttaat ccagattacc agtttacctt
     1921 cttcgatttt catgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     1981 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
     2041 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     2101 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     2161 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     2221 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     2281 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     2341 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     2401 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     2461 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     2521 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     2581 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     2641 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     2701 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     2761 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     2821 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     2881 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     2941 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     3001 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     3061 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     3121 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     3181 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     3241 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     3301 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     3361 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     3421 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     3481 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     3541 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     3601 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     3661 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     3721 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     3781 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc
     3841 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     3901 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     3961 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     4021 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     4081 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     4141 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     4201 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     4261 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     4321 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     4381 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     4441 taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     4501 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     4561 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     4621 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt
     4681 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     4741 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4801 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4861 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4921 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4981 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5041 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5101 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5161 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5221 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5281 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5341 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5401 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5461 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5521 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa
     5581 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     5641 gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     5701 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     5761 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     5821 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     5881 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag
     5941 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     6001 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     6061 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     6121 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     6181 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     6241 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     6301 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     6361 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     6421 tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag
     6481 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     6541 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     6601 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     6661 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     6721 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     6781 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     6841 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     6901 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     6961 cgcttaatgc gccgctacag ggcgcgtccc attcgcca
//
