LOCUS       sequence-182911-        7092 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 7092)
  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 7092)
  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: MSP852
FEATURES             Location/Qualifiers
     source          1..7092
                     /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(870..890)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             complement(927..2027)
                     /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(930..953)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      1061..1070
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             2033..2055
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2070..2094
                     /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(2094..2113)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2095..2113)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2197..2216)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2422..2499
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2500..3582
                     /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="MKPVTLYDVAEYAGVSYQTVSRVVNQDSHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2519..2538)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4391..4582
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4684..4823
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4739..4758
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4838..4855)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5009..5597)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5089..5108)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5708..5727)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5719..6534
                     /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             5719..6534
                     /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(5792..5811)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6626..7081)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6763..6784)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6975..6994
                     /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 cctcaactgg ctgaaaaggc tcttcaggtt ttcacaaaga acacttcatg gcccccaacc
      241 aacattgagt cagcatttaa tcgccaacca ctaacccact gctgcgcctc ctgatttgtc
      301 aacaattcca tgaagttttt cggcacgctt gtgaaataca tagttgtgtt ggtgaaatcc
      361 gctgctgaat gtataccaac aatggcccca tctctagttg atactaatgg actgccacac
      421 tgccccggct tagattgaat ccaatgcttc cagaatatgc catcaaatga agggaatgtg
      481 caactagtgg ctgacaccat gctagacatg ctcttagttt ggaagttggt tgtcacaaga
      541 catatgcgct cttccctttg tggctctcta aatttcagct tttgaggaaa tggagggaaa
      601 tccttaggca tgcgaataat tatcatgtcc ctcccatcaa tgaggtgttg ttgcaaagtc
      661 gtggtgttct tgaccttgaa tacaccatgt agtgattgga ccaacagtgt tccattattt
      721 cttctaaaca agtgcttgtt tgtaatgatg aagggaccaa atccaatacc atacaacgat
      781 gctgtgtgcc catcagattc attcgtcaaa tgacaaatgg tgctcgatat cgggttgtaa
      841 tcacgcggcc ccttaaacaa gctttctcca tgatgatgat gatgatgatg catgtgacca
      901 accagcgggt gtcccgagct cgaattagtc tgcgcgtctt tcagggcttc atcgacagtc
      961 tgacgaccgc tggcggcgtt gatcaccgca gtacgcacgg cataccagaa agcggacatc
     1021 tgcgggatgt tcggcatgat ttcacctttc tgggcgtttt ccatggtggc ggcaatacgt
     1081 ggatctttcg ccaactcttc ctcgtaagac ttcagcgcta cggcacccag cggtttgtct
     1141 ttattaaccg cttccagacc ttcatcagtc agcagatagt tttcgaggaa ctcttttgcc
     1201 agctctttgt tcggactggc ggcgttaata cctgcgctca gcacgccaac gaacggtttg
     1261 gatggttgac ccttgaaggt cggcagtacc gttacaccat aattcacttt gctggtgtcg
     1321 atgttggacc atgcccacgg gccgttgatg gtcatcgctg tttcgccttt attaaaggca
     1381 gcttctgcga tggagtaatc ggtgtctgca ttcatgtgtt tgtttttaat caggtcaacc
     1441 aggaaggtca gacccgcttt cgcgccagcg ttatccacgc ccacgtcttt aatgtcgtac
     1501 ttgccgtttt catacttgaa cgcataaccc ccgtcagcag caatcagcgg ccaggtgaag
     1561 tacggttctt gcaggttgaa catcagcgcg ctcttacctt tcgctttcag ttctttatcc
     1621 agcgccggga tctcttccca ggtttttggc gggttcggca gcagatcttt gttataaatc
     1681 agcgataacg cttcaacagc gatcgggtaa gcaatcagct tgccgttgta acgtacggca
     1741 tcccaggtaa acggatacag cttgtcctgg aacgctttgt ccggggtgat ttcagccaac
     1801 aggccagatt gagcgtagcc accaaagcgg tcgtgtgccc agaagataat gtcagggcca
     1861 tcgccagttg ccgcaacctg tgggaatttc tcttccagtt tatccggatg ctcaacggtg
     1921 actttaattc cggtatcttt ctcgaatttc ttaccgactt cagcgagacc gttatagcct
     1981 ttatcgccgt taatccagat taccagttta ccttcttcga ttttcatgta tatctccttc
     2041 ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata
     2101 gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg
     2161 ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     2221 gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg
     2281 caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg
     2341 cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata
     2401 agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca
     2461 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac
     2521 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggac
     2581 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
     2641 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
     2701 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
     2761 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
     2821 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
     2881 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
     2941 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
     3001 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
     3061 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     3121 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     3181 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     3241 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
     3301 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
     3361 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     3421 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
     3481 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     3541 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     3601 aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc
     3661 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     3721 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga
     3781 ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca
     3841 cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc
     3901 cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac
     3961 ccgactagga tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg
     4021 aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt
     4081 cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt
     4141 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac
     4201 gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag
     4261 ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt
     4321 gagcatcctc tctagtttca tcggtatcat tacccccatg aacagaaatc ccccttacac
     4381 ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag
     4441 ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat
     4501 ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg
     4561 tgatgacggt gaaaacctct gacacatgca gctccaggag acggtcacag cttgtctgta
     4621 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     4681 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     4741 gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg cacagatgcg
     4801 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4861 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4921 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4981 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5041 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5101 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5161 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5221 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5281 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5341 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5401 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     5461 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5521 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5581 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5641 acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac
     5701 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga
     5761 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     5821 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
     5881 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     5941 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     6001 tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct
     6061 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     6121 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     6181 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     6241 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     6301 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     6361 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     6421 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     6481 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta
     6541 attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6601 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     6661 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     6721 tataaatcaa aagaatagac cgaaataggg ttgagtgttg ttccagtttg gaacaagagt
     6781 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     6841 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     6901 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     6961 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     7021 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     7081 tcccattcgc ca
//
