LOCUS       sequence-329621-        6539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses BoNT/X-LC(1-439)-6xHis in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6539)
  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 6539)
  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/X
FEATURES             Location/Qualifiers
     source          1..6539
                     /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"
     RBS             1480..1502
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1517..1541
                     /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(1541..1560)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1542..1560)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1644..1663)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1869..1946
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1947..3029
                     /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(1966..1985)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3838..4029
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4023..4045)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4131..4270
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4186..4205
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4285..4302)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4456..5044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4536..4555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5155..5174)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5166..5981
                     /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             5166..5981
                     /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(5239..5258)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6073..6528)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6210..6231)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6422..6441
                     /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 ggtggtggtt ttttgagttg cggtaaatgg
      181 cattatacaa caacccgtta cgtggacaaa ttttaataaa cgcacgaagt gcgttggact
      241 cgatcttttc aaaataagac gactctaaca gctggccttg gaaatcgttc gacccctggg
      301 agctgatatt aaaaccctct aaagtgctgt aactattgtc gtccaggatg tttacataga
      361 tagggtcgat tggacgctcc ttaaggtagt gcttgcgcac aaggatggag aaacgctgtg
      421 ccaagttaga ctcattaagt acaaacaaga tggtattaag tttcttctcg aactcagctg
      481 tatcaagttt gaagttaccc aaacgacctt gtactgggat tttgtttttg atatatttta
      541 acaaatcatt ctctacggta acagtattaa gacgctctga gatcagagta gtatagttgt
      601 ttttggccgt ttcgatgatc tttttgatga tcaatgatga aattgcttta gagtcaattc
      661 caccaaacgt aagaagctcc tcaaagataa gactgttttg ttgacgcgaa gtttcaatct
      721 ttcctgtgtc aaaattatag taaaagttgc gattgctgat gccgtaaaga ttatgagtca
      781 cgtgaaccag ctcgtgcatc aatgtcgaag caggatctgg agcgaactca cgttttacga
      841 atttatttac aatattcacc aaactacggt agttgccgta gctctcatcg aatggtttca
      901 agtagaatgg ggaaaacgac acttcactca gtgtcccttc accgttagag atcggcgtag
      961 aatacaatcc gtatgttgca ttattcgcaa tatcggggcc gggcccataa atgaccagat
     1021 tagcttgcag gttagacacg atgttgttat tctcttgata cgcgattgtc tcattgtccg
     1081 acagggttaa tgcgccgttg gagacaagag gcaatgggat agaggaggaa atcaactcta
     1141 aaagcttctc tccctcaggt ttgctcttga tacgctccag caccttgatt acaccttgca
     1201 agaactcgtc cttttcactc ggagtgttca ggtaattggg attatagata gcatccgcct
     1261 ccatgatagg ctctgacgga atgtttaaat cgtttgtgtt attcgtaaag ttgtaacgtt
     1321 ccggaacgat ccagatgttt ttaatcactt ggaaagcttt gaaaggcccc ttgcccttgt
     1381 tgattttatc agaatggcgg ggggggcgca tcgtgattac gttgatacca tcaatgggat
     1441 cgttgtagtt aaacttgtta atttcaagtt tcatgtatat ctccttctta aagttaaaca
     1501 aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta
     1561 atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg
     1621 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc
     1681 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
     1741 cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa
     1801 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg
     1861 agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     1921 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     1981 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     2041 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     2101 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     2161 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     2221 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     2281 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     2341 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     2401 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     2461 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     2521 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     2581 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     2641 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     2701 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     2761 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     2821 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     2881 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     2941 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     3001 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     3061 cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc
     3121 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
     3181 ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg
     3241 agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa
     3301 gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc
     3361 atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     3421 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     3481 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     3541 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     3601 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     3661 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     3721 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     3781 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     3841 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     3901 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     3961 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     4021 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     4081 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     4141 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     4201 ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     4261 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4321 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4381 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4441 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4501 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4561 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4621 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4681 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4741 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4801 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4861 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     4921 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4981 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5041 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5101 ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     5161 gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca
     5221 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     5281 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     5341 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     5401 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     5461 ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     5521 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     5581 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     5641 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     5701 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     5761 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     5821 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     5881 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     5941 ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt catgagcgga
     6001 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6061 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     6121 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     6181 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     6241 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     6301 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     6361 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     6421 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     6481 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
