LOCUS       sequence-346778-        6455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses BoNT/E-LC(1-411)-6xHis in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6455)
  AUTHORS   Blum TR, Liu H, Packer MS, Xiong X, Lee PG, Zhang S, Richter M, 
            Minasov G, Satchell KJF, Dong M, Liu DR
  TITLE     Phage-assisted evolution of botulinum neurotoxin proteases with 
            reprogrammed specificity.
  JOURNAL   Science. 2021 Feb 19;371(6531):803-810. doi: 
            10.1126/science.abf5972.
  PUBMED    33602850
REFERENCE   2  (bases 1 to 6455)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2021-02-19- 19"; volume: "371"; 
            issue: "6531"; pages: "803-810"
COMMENT     Sequence Label: pET-BoNT/E
FEATURES             Location/Qualifiers
     source          1..6455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(19..66)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     62..80
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(133..150)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1389..1411
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1426..1450
                     /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(1450..1469)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1451..1469)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1553..1572)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1778..1855
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1856..2938
                     /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(1875..1894)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3747..3938
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3932..3954)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4040..4179
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4095..4114
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4194..4211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4365..4953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4445..4464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5064..5083)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5075..5890
                     /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             5075..5890
                     /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(5148..5167)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5982..6437)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6119..6140)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6331..6350
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta
       61 tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc
      121 agccggatct cagtggtggt ggtggtggtg aaaccggatt attttcttca ctaacccccg
      181 tcctgtgatg ggtttaatga tgcgcggatt taggttggcg ttttggcctc ggaaattcac
      241 ctttaggtta tttatattgt aaccctcact aatgttgtat atggaatcat tcaggagatt
      301 cgacagcttg aaatacttat actgtccgat gtatgtctct cggcatttga cctggaattt
      361 ggtggccaag tcgaactccg tgaagctgta aagtttcttc aaaatatcgt cgaatttgtt
      421 aatgtttacc gaatagattc ctgacgcgtc tttgtctaat ccatatttct cttggaatat
      481 gtccttatac ggattcagtt gagggtttga gacttgaacc ttggataact tagaagcgat
      541 ttttctataa tcatttagta agttggtata gatgtcattg tactgtgcga cagttataat
      601 gttcagatcg ttgcctccaa atgtaagaaa ttcctcgatg ttaatgccct tcctgttcgt
      661 tatcaatggg ttctgttgct gagtaatgat acacgtagtg gttatcccct tagcgccata
      721 caggccgtgt aaagagtgta tgagttcgtg cataagcgtg agtgcggggt cttggataaa
      781 ctcatttata ctattatcat tgaagcgaaa actgtattcg ggagaaaaag taacgatcgc
      841 tatgctaccg aacccatggt ttgacggcat atagttattt ctcaggctaa tattactcga
      901 atttgtttca aataaatctg gttccgcccc cattataata acgttgggca agagaatatg
      961 ctgtgatcca ttgctaaatt tgatttccac ggcggatgca tcgcctatat gaaactggtt
     1021 gtctggtgta ttatcgttac ctaggtaagg attagcttta ctaagctctt caagcaaaat
     1081 acctccggat agattgttat taatcctgtt aaagattttg gttactattt taaggaatct
     1141 gtccttctct tcatccgact ggagataatt ggggtcgtaa taggaagagt caccgttctt
     1201 caacgaagta ggtggatgaa aatcttgcgg tgtcgtacca ataacgttac gctcaggtat
     1261 aatccatata ttcttcataa tattgaaaga tttgtagaac tcttgacacc cacccggctt
     1321 tatgtatagg atggtacgat cgtttacagg gtcattgtaa ttaaagctgt tgatttttgg
     1381 catgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc
     1441 tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctac
     1501 gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc
     1561 gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc
     1621 ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat
     1681 gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta
     1741 atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac
     1801 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     1861 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     1921 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     1981 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     2041 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     2101 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     2161 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     2221 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     2281 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
     2341 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     2401 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     2461 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     2521 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     2581 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     2641 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     2701 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     2761 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     2821 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     2881 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     2941 gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct
     3001 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     3061 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg
     3121 tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg
     3181 tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt
     3241 tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc
     3301 tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat
     3361 caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa
     3421 caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc
     3481 cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac
     3541 ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc
     3601 gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat
     3661 cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca
     3721 gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg
     3781 cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg
     3841 atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct
     3901 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     3961 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     4021 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
     4081 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa
     4141 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     4201 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4261 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4321 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4381 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4441 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4501 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4561 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4621 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4681 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4741 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4801 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4861 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4921 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4981 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact
     5041 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     5101 ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     5161 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
     5221 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     5281 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     5341 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
     5401 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     5461 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     5521 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     5581 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     5641 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     5701 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     5761 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     5821 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     5881 gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac
     5941 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     6001 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     6061 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     6121 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     6181 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     6241 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     6301 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     6361 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     6421 gccgctacag ggcgcgtccc attcgccaat ccgga
//
