LOCUS       sequence-329620-        7619 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses MBP-F(3230)A3-6xHis in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7619)
  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 7619)
  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-mF(3230)A3
FEATURES             Location/Qualifiers
     source          1..7619
                     /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(1454..2554)
                     /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(1457..1480)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      1588..1597
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             2560..2582
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2597..2621
                     /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(2621..2640)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2622..2640)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2949..3026
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3027..4109
                     /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(3046..3065)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4918..5109
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5103..5125)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5211..5350
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5266..5285
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5365..5382)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5536..6124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5616..5635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6235..6254)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6246..7061
                     /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             6246..7061
                     /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(6319..6338)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(7153..7608)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7290..7311)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7502..7521
                     /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 ggtggtgcga ttttctcaac caggccttgt
      181 ctgggatgga gtcaatgatt ttcagattca gtttgatgtt ttgaccacga ttattcacgg
      241 ccagatggcc gatgttgaag ccttcgctga cggtatagat gtcatcgtcc atcaggttcg
      301 ggactttcag gaagccattt ttgatgaagt aggtattacg acatttgact ttgagcttgt
      361 tcgccagatc gatttcggtg aagccgtaca gtttcttgta gatttcattg aatttgtttt
      421 cattcacggt ataagaacca tccgcatttt tgtccaggcc atatttccat tgaaagtagt
      481 ctttgtattc attgatgtcg tattccggcg gagcggagtt aacacgagac aagcgcgttg
      541 caatcttttc atagttggcc agcaaattgt tatagatttt ctctttcatg gccgaggtaa
      601 tgatgttcaa atcttgaccg ccgaaggtca ggaactcttc caggcgaatc ggcttttccg
      661 caatcatcag aggtgcctgc ttaaccttga tagtctcttt atacgtcaca ccaagggcac
      721 cgtacaagcc atgcaatgaa tggatcagtt cgtgtgccag gatgatcgcc gggtcggcga
      781 tgaagctctc ggtagaagaa ttgtaaccac cgctgatatc attgaaggtg tacccgtact
      841 ccgggctaaa cgtcacgata ttgatgctgc caaaaccgtc atggctcggg tcataaacac
      901 cgtcgctatc catcagttta cgcaccgggt agatatagtt ttcgaagatg tccggaccgg
      961 gacccagaac cagcaggttc aggacaatgt tagatttcac gttggtcgag gacttgatgt
     1021 taacgctggt agtgcgggta accgggtgaa actcgttaat aggcgtatgc tcattaccca
     1081 gataaggctt tgcgtagcta atctcttgca gcagggcctc acctgccgga ttgctgttaa
     1141 tgcgtttgaa cagcttgatg gtggttttca aataacgatc tttctccgca tcggtggtca
     1201 ggtaattcgg gtcgtaatgt gcggacaaac cgttttccaa agacgcaggt ggatcgaagt
     1261 cgcttgggtc agtaccaatc gtattacgct ccggaataat ccacacattg cgcatgattt
     1321 caaaggcctt atagtacttc ttgctcttct cttcatacgg gatctgcata tacaggatcg
     1381 tgtcgtcgtt tactgggtcg ttgtagttga agctgttgat gaccaccggc atagaaccac
     1441 ccgaaccacc attagtctgc gcgtctttca gggcttcatc gacagtctga cgaccgctgg
     1501 cggcgttgat caccgcagta cgcacggcat accagaaagc ggacatctgc gggatgttcg
     1561 gcatgatttc acctttctgg gcgttttcca tggtggcggc aatacgtgga tctttcgcca
     1621 actcttcctc gtaagacttc agcgctacgg cacccagcgg tttgtcttta ttaaccgctt
     1681 ccagaccttc atcagtcagc agatagtttt cgaggaactc ttttgccagc tctttgttcg
     1741 gactggcggc gttaatacct gcgctcagca cgccaacgaa cggtttggat ggttgaccct
     1801 tgaaggtcgg cagtaccgtt acaccataat tcactttgct ggtgtcgatg ttggaccatg
     1861 cccacgggcc gttgatggtc atcgctgttt cgcctttatt aaaggcagct tctgcgatgg
     1921 agtaatcggt gtctgcattc atgtgtttgt ttttaatcag gtcaaccagg aaggtcagac
     1981 ccgctttcgc gccagcgtta tccacgccca cgtctttaat gtcgtacttg ccgttttcat
     2041 acttgaacgc ataacccccg tcagcagcaa tcagcggcca ggtgaagtac ggttcttgca
     2101 ggttgaacat cagcgcgctc ttacctttcg ctttcagttc tttatccagc gccgggatct
     2161 cttcccaggt ttttggcggg ttcggcagca gatctttgtt ataaatcagc gataacgctt
     2221 caacagcgat cgggtaagca atcagcttgc cgttgtaacg tacggcatcc caggtaaacg
     2281 gatacagctt gtcctggaac gctttgtccg gggtgatttc agccaacagg ccagattgag
     2341 cgtagccacc aaagcggtcg tgtgcccaga agataatgtc agggccatcg ccagttgccg
     2401 caacctgtgg gaatttctct tccagtttat ccggatgctc aacggtgact ttaattccgg
     2461 tatctttctc gaatttctta ccgacttcag cgagaccgtt atagccttta tcgccgttaa
     2521 tccagattac cagtttacct tcttcgattt tcatgtatat ctccttctta aagttaaaca
     2581 aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta
     2641 atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg
     2701 cgccacaggt gcggttgctg gcgcctatat cgccgacatc atcgatgggg aagatcgggc
     2761 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
     2821 cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa
     2881 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg
     2941 agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     3001 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     3061 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     3121 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     3181 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     3241 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     3301 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     3361 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     3421 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     3481 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     3541 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     3601 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     3661 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     3721 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     3781 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     3841 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     3901 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     3961 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     4021 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     4081 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     4141 cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc
     4201 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
     4261 ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg
     4321 agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa
     4381 gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc
     4441 atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     4501 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     4561 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     4621 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     4681 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     4741 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     4801 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     4861 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     4921 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     4981 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     5041 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     5101 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     5161 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     5221 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     5281 ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     5341 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5401 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5461 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5521 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5581 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5641 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5701 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5761 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5821 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5881 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5941 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     6001 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6061 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6121 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6181 ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     6241 gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca
     6301 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     6361 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     6421 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     6481 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     6541 ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     6601 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     6661 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     6721 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     6781 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     6841 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     6901 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     6961 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     7021 ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt catgagcgga
     7081 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7141 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     7201 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     7261 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     7321 acgtggactc caacgtcaaa ggacgaaaaa ccgtctatca gggcgatggc ccactacgtg
     7381 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     7441 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     7501 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     7561 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
