LOCUS       sequence-329589-        6455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E(4130)A2-6xHis 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-E(4130)A2
FEATURES             Location/Qualifiers
     source          1..6455
                     /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             1396..1418
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1433..1457
                     /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(1457..1476)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1458..1476)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1560..1579)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1785..1862
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1863..2945
                     /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(1882..1901)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3754..3945
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3939..3961)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4047..4186
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4102..4121
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4201..4218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4372..4960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4452..4471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5071..5090)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5082..5897
                     /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             5082..5897
                     /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(5155..5174)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5989..6444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6126..6147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6338..6357
                     /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 ggtggtgaaa ccggattatt ttcttcacta
      181 acccccgtcc tgtgatgggt ttaatgatac gcggatttag gttggcgttt tggcctcgga
      241 aattcacctt taggttattt atattgtaac cctcactaat gttgtatatg gaatcattca
      301 ggagattcga cagcttaaaa aacttaggcc gtccgatgta tgtctctcgg catttgacct
      361 ggaatttggt ggccaagtcg aactccgtga agctgtaaag tttcttcaaa atatcgtcga
      421 atttgttaat gtttaccgaa tagattcctg acgcgtcttt gtctaatcca tatttttctt
      481 ggaatatgtc cttatacgga ttcagttgag ggtttgagac ttgaaccttg gataacttag
      541 aagcgatttt tctataatca tttagtaagt tggtatagat gtcattgtac tgtgcgacag
      601 ttataatgtt cagatcgttg cctccaaatg taagaaattc ctcgatttta atgcccttcc
      661 tgttcgttat caatgggttc tgttgctgag taatggtaca cgtagtggtt atccccttag
      721 cgccatacag gccgtgtaaa gagtgtatga gttcgtgcat aagcgtgagt gcggggtctt
      781 ggataaactc atttatacta ttatcattga agcgaaaact gtattcggga gaaaaagtaa
      841 cgattgctat gctaccgaac ccatggtttg acggcttata gttatttctc aggctaatat
      901 ttctctgata tgtcaaaaat aaatttggtt ccgcccccat tataataacg ttgggcaaga
      961 gaatatgctg tgatccattg ctaaatttga tttccacggc ggatgcatcg cctatatgaa
     1021 actggttgtc tggtgtatca tcgttaccta ggtaaggatt agctttacta agctcttcaa
     1081 gcaaaatact tccggctaga ttgttattaa tcctgttaaa gattttggtt actattttaa
     1141 ggaatctgtc cttctcttca tccgactgga gataattggg gtcgtaatag gaagagtcac
     1201 cgttcttcaa cgaagtaggt ggatgaaaat cttgcggtgt cgtaccaata acgttacgct
     1261 caggtataat ccatatattc ttcataatat tgaaagattt gtagaactcg tgatacccac
     1321 ccggctttat gtataggatg gtacgatcgt ttacagggtc attgtaatta aagctgttga
     1381 tttttggcat gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
     1441 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat
     1501 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
     1561 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
     1621 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
     1681 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
     1741 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
     1801 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     1861 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     1921 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     1981 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     2041 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     2101 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     2161 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     2221 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     2281 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     2341 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     2401 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     2461 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     2521 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     2581 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     2641 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     2701 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     2761 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     2821 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     2881 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2941 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
     3001 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     3061 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     3121 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
     3181 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc
     3241 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg
     3301 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
     3361 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
     3421 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
     3481 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
     3541 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
     3601 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
     3661 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
     3721 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
     3781 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
     3841 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
     3901 cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3961 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     4021 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     4081 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg
     4141 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     4201 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4261 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4321 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4381 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4441 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4501 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4561 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4621 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4681 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4741 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4801 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4861 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4921 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4981 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa
     5041 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     5101 aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     5161 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc
     5221 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     5281 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     5341 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat
     5401 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     5461 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     5521 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     5581 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     5641 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     5701 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     5761 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     5821 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     5881 tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa
     5941 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     6001 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     6061 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     6121 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     6181 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     6241 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     6301 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     6361 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     6421 ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
