LOCUS       sequence-329557-        7628 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses MBP-BoNT/F-LC(1-428)-6xHis in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7628)
  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 7628)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-mBoNT/F
FEATURES             Location/Qualifiers
     source          1..7628
                     /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(1463..2563)
                     /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(1466..1489)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      1597..1606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             2569..2591
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2606..2630
                     /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(2630..2649)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2631..2649)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2733..2752)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2958..3035
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3036..4118
                     /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(3055..3074)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4927..5118
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5112..5134)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5220..5359
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5275..5294
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5374..5391)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5545..6133)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5625..5644)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6244..6263)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6255..7070
                     /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             6255..7070
                     /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(6328..6347)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(7162..7617)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7299..7320)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7511..7530
                     /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 ggtggtggaa cttcacgatt ttctcaacca
      181 ggcccttgtc tgggatggag tcaatgattt tcggattcag tttgatgttc tgaccacgat
      241 tattcacggc cagattgccg atgttgaagc cttcgctgac ggtatagatg tcatcgtcca
      301 gcaggttcgg gactttcagg aagccatatt tgatgaagta ggtattacga catttgactt
      361 tgaacttgtt cgccagatcg atttcggtga agctgtacag tttcttgtag atttcattga
      421 atttgttttc attcacggta taagaaccat ccgcattttt gtccaggcca tatttccatt
      481 gaaagtagtc tttgtactca ttaatgtcgt attccggcgg agcggagtta acacgagaca
      541 agcgcgttgc aatcttttca tagttggcca gcaaattgtt atagattttc tctttcatgg
      601 ccgaggtaat gatgttcaaa tcttgaccgc cgaaggtcag gaactcttcc aggcgaatcg
      661 gcttttccgc aatcatcaga ggtgcctgct taaccttgat agtctcttta tacgtcacac
      721 cacgggcacc gtacaagcca tgcaatgcat ggatcagttc gtgtgccagg ctgatcgccg
      781 ggtcggcgat gaagctctcg gtagaagaat tgtaaccacc gctgatatca ttgaaggtgt
      841 actcgtactc cgggctaaac gtcacgatat tgatgctgcc aaaaccgtca ttgctcgggt
      901 cataaacacc gccgctatcc atcagtttac gcaccgggta gctagagttt tcgaagatgt
      961 ccggaccggc acccagaacc agcaggttca ggataatgct agatttcacg ttggtcgagg
     1021 acttgatgtt aacgctggta gtgcgggtaa ccgggtgaaa ctcgttaata ggcgtatgct
     1081 cattacccag ataaggcttt gcgtagctaa tctcttgcag caggacctca cctgccggat
     1141 tgctgttaat gcgtttgaac agcttgatgg tggttttcaa ataacgatct ttctccgcat
     1201 cggtggtcag gtaattcggg tcgtaatatg cggacgagcc gttttccaaa gacgcaggtg
     1261 gatcgaagtc gcttgggtca gtaccaatcg tattacgctc cggaataatc cacacattgc
     1321 gcatgatttc aaaggcctta tagtacttct tgctcttctc ttcatacggg atttgcatat
     1381 acaggatcgt gtcgtcgttt actgggtcgt tgtagttgaa gctgttgatg accaccggca
     1441 tagaaccacc cgaaccacca ttagtctgcg cgtctttcag ggcttcatcg acagtctgac
     1501 gaccgctggc ggcgttgatc accgcagtac gcacggcata ccagaaagcg gacatctgcg
     1561 ggatgttcgg catgatttca cctttctggg cgttttccat ggtggcggca atacgtggat
     1621 ctttcgccaa ctcttcctcg taagacttca gcgctacggc acccagcggt ttgtctttat
     1681 taaccgcttc cagaccttca tcagtcagca gatagttttc gaggaactct tttgccagct
     1741 ctttgttcgg actggcggcg ttaatacctg cgctcagcac gccaacgaac ggtttggatg
     1801 gttgaccctt gaaggtcggc agtaccgtta caccataatt cactttgctg gtgtcgatgt
     1861 tggaccatgc ccacgggccg ttgatggtca tcgctgtttc gcctttatta aaggcagctt
     1921 ctgcgatgga gtaatcggtg tctgcattca tgtgtttgtt tttaatcagg tcaaccagga
     1981 aggtcagacc cgctttcgcg ccagcgttat ccacgcccac gtctttaatg tcgtacttgc
     2041 cgttttcata cttgaacgca taacccccgt cagcagcaat cagcggccag gtgaagtacg
     2101 gttcttgcag gttgaacatc agcgcgctct tacctttcgc tttcagttct ttatccagcg
     2161 ccgggatctc ttcccaggtt tttggcgggt tcggcagcag atctttgtta taaatcagcg
     2221 ataacgcttc aacagcgatc gggtaagcaa tcagcttgcc gttgtaacgt acggcatccc
     2281 aggtaaacgg atacagcttg tcctggaacg ctttgtccgg ggtgatttca gccaacaggc
     2341 cagattgagc gtagccacca aagcggtcgt gtgcccagaa gataatgtca gggccatcgc
     2401 cagttgccgc aacctgtggg aatttctctt ccagtttatc cggatgctca acggtgactt
     2461 taattccggt atctttctcg aatttcttac cgacttcagc gagaccgtta tagcctttat
     2521 cgccgttaat ccagattacc agtttacctt cttcgatttt catgtatatc tccttcttaa
     2581 agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga
     2641 gtcgtattaa tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg
     2701 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
     2761 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg
     2821 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
     2881 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     2941 agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     3001 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     3061 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     3121 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     3181 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     3241 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     3301 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     3361 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     3421 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     3481 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     3541 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     3601 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     3661 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     3721 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     3781 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     3841 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     3901 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     3961 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     4021 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     4081 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     4141 tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc
     4201 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     4261 atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc
     4321 tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc
     4381 ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt
     4441 atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     4501 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     4561 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     4621 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg
     4681 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     4741 cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
     4801 ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc
     4861 atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag
     4921 gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag
     4981 acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt
     5041 gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat
     5101 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     5161 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     5221 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
     5281 cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag
     5341 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5401 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5461 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5521 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5581 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5641 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5701 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5761 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5821 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5881 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5941 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     6001 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6061 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6121 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6181 aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta
     6241 atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa
     6301 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     6361 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
     6421 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     6481 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     6541 tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt
     6601 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     6661 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     6721 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     6781 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     6841 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     6901 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     6961 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     7021 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc
     7081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7141 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     7201 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     7261 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     7321 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     7381 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     7441 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     7501 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     7561 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     7621 attcgcca
//
