LOCUS       sequence-331960-        9391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  negative selection PACE Accessory Plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9391)
  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 9391)
  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: APneg-VAMP1d
FEATURES             Location/Qualifiers
     source          1..9391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(106..123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(240..785)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(320..339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1409..1428
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1409..1427
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1676..1693
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2704..3783
                     /codon_start=1
                     /gene="luxA"
                     /product="LuxA luciferase subunit"
                     /label=LuxA
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHH
                     FTEFGLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQMSKGRFRFG
                     ICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYMEADNEHIKFHKVKVNPAAY
                     SRGGAPVYVVAESASTTEWAAQFGLPMILSWIINTNEKKAQLELYNEVAQEYGHDIHNI
                     DHCLSYITSVDHDSIKAKEICRKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFV
                     LKGHKDTNRRIDYSYEINPVGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASM
                     KLFQSDVMPFLKEKQRSLLY"
     CDS             3847..4827
                     /codon_start=1
                     /gene="luxB"
                     /product="LuxB luciferase subunit"
                     /label=LuxB
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="KFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHF
                     SDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVRIAEEACLLDQLSEGRFILGF
                     SDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYT
                     PGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEID
                     HQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITA
                     AKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIVDDNIKKYHTEYT"
     CDS             5109..5558
                     /codon_start=1
                     /product="lysozyme from bacteriophage T7"
                     /label=T7 lysozyme
                     /note="inhibitor of T7 RNA polymerase"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAVLRAHHEVAPKASPSFDLKRWWEKNELVTSDR"
     CDS             join(8627..9391,1..24)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 gagatcacca aggtagtcgg caaaacggat cgcttcatgt ggcagtagaa aaaagactgc
       61 accggtgcgt cagcagaata tgtgatacag gatatattcc gcttcctcgc tcactgactc
      121 gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcggagattt
      181 cctggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc aaagccgttt
      241 ttccataggc tccgcccccc tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg
      301 cgaaacccga caggactata aagataccag gcgtttcccc ctggcggctc cctcgtgcgc
      361 tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct
      421 cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca
      481 cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      541 cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt
      601 tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg
      661 ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac
      721 cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat
      781 ctcaagaaga tcatcttatt aaggggtctg acgctcagtg gaacgaaaac tcacgttaag
      841 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
      901 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
      961 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     1021 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     1081 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     1141 attcgagctc gcccggggat cgaccagttg gtgattttga acttttgctt tgccacggaa
     1201 cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca actcagcaaa agttcgattt
     1261 attcaacaaa gccgccgtcc cgtcaagtca gcgtaatgct ctgccagtgt tacaaccaat
     1321 taacaagagg acatccggtc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1381 ttttgctgag gagacttagg gaccctacta atacgactca ctatagggag aaagaaggag
     1441 cgacattgct ccgtgtattc actcgttgga atgaatacac agtgcagtgt ttattctgtt
     1501 atttatgcca aaaataaagg ccactatcag gcagctttgt tgttctgttt accaagttca
     1561 ggaggtaact cataagaaag acctgcaggt gcagtaaaaa aaggaaaaat tgaaaaaatt
     1621 attattcgca attcctttag ttgttccttt ctattctcac tccgctgaaa ctgttcatca
     1681 ccatcaccat cacgctgaaa ctgttgaaag ttgtttagca aaaccccata cagaaaattc
     1741 atttactaac gtctggaaag acgacaaaac tttagatcgt tacgctaact atgagggctg
     1801 tctgtggaat gctacaggcg ttgtagtttg tactggtgac gaaactcagt gttacggtac
     1861 atgggttcct attgggcttg ctatccctga aaatgagggt ggtggctctg agggtggcgg
     1921 ttctgagggt ggcggttctg agggtggcgg tactaaacct cctgagtacg gtgatacacc
     1981 tattccgggc tatacttata tcaaccctct cgacggcact tatccgcctg gtactgagca
     2041 aaaccccgct aatcctaatc cttctcttga ggagtctcag cctcttaata ctttcatgtt
     2101 tcagaataat aggttccgaa ataggcaggg ggcattaact gtttatacgg gcactgttac
     2161 tcaaggcact gaccccgtta aaacttatta ccagtacact cctgtatcat caaaagccat
     2221 gtatgacgct tactggaacg gtaaattcag agactgcgct ttccattctg gctttaatga
     2281 ggatccattc gtttgtgaat atcaaggcca atcgtctgac ctgcctcaac ctcctgtcaa
     2341 tgctggcggc ggctctggtg gtggttctgg tggcggctct gagggtggtg gctctgaggg
     2401 tggcggttct gagggtggcg gctctgaggg aggcggttcc ggtggtggct cttcccaaat
     2461 ggctcaagtc ggtgacggtg ataattcacc tttaatgaat aatttccgtc aatatttacc
     2521 ttccctccct caatcggttg aatgtcgccc ttttgtcttt ggcgctggta aaccttacga
     2581 gttcagtatc gactgcgata agatcaacct gttccgcggt gtctttgcgt ttcttttata
     2641 tgttgccacc tttatgtatg tattttctac gtttgctaac atactgcgta ataaggagtc
     2701 ttaatgaaat ttggaaactt tttgcttaca taccaacctc cccaattttc ccaaacagag
     2761 gtaatgaaac gtttggttaa attaggtcgc atctctgagg agtgtggttt tgataccgta
     2821 tggttactgg agcatcattt cacggagttt ggtttgcttg gtaaccctta tgtcgctgct
     2881 gcatatttac ttggcgcgac taaaaaattg aatgtaggaa ctgccgctat tgttcttccc
     2941 acagcccatc cagtacgcca acttgaagat gtgaatttat tggatcaaat gtcaaaagga
     3001 cgatttcggt ttggtatttg ccgagggctt tacaacaagg actttcgcgt attcggcaca
     3061 gatatgaata acagtcgcgc cttagcggaa tgctggtacg ggctgataaa gaatggcatg
     3121 acagagggat atatggaagc tgataatgaa catatcaagt tccataaggt aaaagtaaac
     3181 cccgcggcgt atagcagagg tggcgcaccg gtttatgtgg tggctgaatc agcttcgacg
     3241 actgagtggg ctgctcaatt tggcctaccg atgatattaa gttggattat aaatactaac
     3301 gaaaagaaag cacaacttga gctttataat gaagtggctc aagaatatgg gcacgatatt
     3361 cataatatcg accattgctt atcatatata acatctgtag atcatgactc aattaaagcg
     3421 aaagagattt gccggaaatt tctggggcat tggtatgatt cttatgtgaa tgctacgact
     3481 atttttgatg attcagacca aacaagaggt tatgatttca ataaagggca gtggcgtgac
     3541 tttgtattaa aaggacataa agatactaat cgccgtattg attacagtta cgaaatcaat
     3601 cccgtgggaa cgccgcagga atgtattgac ataattcaaa aagacattga tgctacagga
     3661 atatcaaata tttgttgtgg atttgaagct aatggaacag tagacgaaat tattgcttcc
     3721 atgaagctct tccagtctga tgtcatgcca tttcttaaag aaaaacaacg ttcgctatta
     3781 tattatggcg gtggcggtag cggcggtggc ggtagcggcg gtggcggtag cggcggtggc
     3841 ggtagcaaat ttggattgtt cttccttaac ttcatcaatt caacaactgt tcaagaacag
     3901 agtatagttc gcatgcagga aataacggag tatgttgata agttgaattt tgaacagatt
     3961 ttagtgtatg aaaatcattt ttcagataat ggtgttgtcg gcgctcctct gactgtttct
     4021 ggttttctgc tcggtttaac agagaaaatt aaaattggtt cattaaatca catcattaca
     4081 actcatcatc ctgtccgcat agcggaggaa gcttgcttat tggatcagtt aagtgaaggg
     4141 agatttattt tagggtttag tgattgcgaa aaaaaagatg aaatgcattt ttttaatcgc
     4201 ccggttgaat atcaacagca actatttgaa gagtgttatg aaatcattaa cgatgcttta
     4261 acaacaggct attgtaatcc agataacgat ttttatagct tccctaaaat atctgtaaat
     4321 ccccatgctt atacgccagg cggacctcgg aaatatgtaa cagcaaccag tcatcatatt
     4381 gttgagtggg cggccaaaaa aggtattcct ctcatcttta agtgggatga ttctaatgat
     4441 gttagatatg aatatgctga aagatataaa gccgttgcgg ataaatatga cgttgaccta
     4501 tcagagatag accatcagtt aatgatatta gttaactata acgaagatag taataaagct
     4561 aaacaagaga cgcgtgcatt tattagtgat tatgttcttg aaatgcaccc taatgaaaat
     4621 ttcgaaaata aacttgaaga aataattgca gaaaacgctg tcggaaatta tacggagtgt
     4681 ataactgcgg ctaagttggc aattgaaaag tgtggtgcga aaagtgtatt gctgtccttt
     4741 gaaccaatga atgatttgat gagccaaaaa aatgtaatca atattgttga tgataatatt
     4801 aagaagtacc acacggaata tacctaaact taattaacgg cactcctcag caaatataat
     4861 gaccctcttg ataacccaag agggcatttt ttaatgccca tggcgtcaat tccgatttcc
     4921 ggtattctag agcacagcta acaccacgtc gtccctatct gctgccctag gtctatgagt
     4981 ggttgctgga taactttacg ggcatgcata aggctcgtat aatatattca gggagaccac
     5041 aacggtttcc ctctacaaat aattttgttt aacttttact agagttcctt cctcgaaagg
     5101 aaaaaaaaat ggctcgtgta cagtttaaac aacgtgaatc tactgacgca atctttgttc
     5161 actgctcggc taccaagcca agtcagaatg ttggtgtccg tgagattcgc cagtggcaca
     5221 aagagcaggg ttggctcgat gtgggatacc actttatcat caagcgagac ggtactgtgg
     5281 aggcaggacg agatgagatg gctgtaggct ctcacgctaa gggttacaac cacaactcta
     5341 tcggcgtctg ccttgttggt ggtatcgacg ataaaggtaa gttcgacgct aactttacgc
     5401 cagcccaaat gcaatccctt cgctcactgc ttgtcacact gctggctaag tacgaaggcg
     5461 ctgtgcttcg cgcccatcat gaggtggcgc cgaaggcttc cccttcgttc gaccttaagc
     5521 gttggtggga gaagaacgaa ctggtcacat ctgaccgtac ctctaaccgt cgtctgcagc
     5581 agacccaggc tcaggttgaa gaagttgttg acatcatccg tgttaacgtt gacaaagttc
     5641 tggaacgtga ccagaaactg tctgaactgg acgaccgtgc tgacgctctg caggctggtg
     5701 cttctcagtt cgaatcttct gctgctaaac tgaaacgtaa cacgattaac atcgctaaga
     5761 acgacttctc tgacatcgaa ctggctgcta tcccgttcaa cactctggct gaccattacg
     5821 gtgagcgttt agctcgcgaa cagttggccc ttgagcatga gtcttacgag atgggtgaag
     5881 cacgcttccg caagatgttt gagcgtcaac ttaaagctgg tgaggttgcg gataacgctg
     5941 ccgctaagcc tctcatcact accctactcc ctaagatgat tgcacgcatc aacgactggt
     6001 ttgaggaagt gaaagctaag cgcggcaagc gcccgacagc cttccagttc ctgcaagaaa
     6061 tcaagccgga agccgtagcg tacatcacca ttaagaccac tctggcttgc ctaaccagtg
     6121 ctgacaatac aaccgttcag gctgtagcaa gcgcaatcgg tcgggccatt gaggacgagg
     6181 ctcgcttcgg tcgtatccgt gaccttgaag ctaagcactt caagaaaaac gttgaggaac
     6241 aactcaacaa gcgcgtaggg cacgtctaca agaaagcatt tatgcaagtt gtcgaggctg
     6301 acatgctctc taagggtcta ctcggtggcg aggcgtggtc ttcgtggcat aaggaagact
     6361 ctattcatgt aggagtacgc tgcatcgaga tgctcattga gtcaaccgga atggttagct
     6421 tacaccgcca aaatgctggc gtagtaggtc aagactctga gactatcgaa ctcgcacctg
     6481 aatacgctga ggctatcgca acccgtgcag gtgcgctggc tggcatctct ccgatgttcc
     6541 aaccttgcgt agttcctcct aagccgtgga ctggcattac tggtggtggc tattgggcta
     6601 acggtcgtcg tcctctggcg ctggtgcgta ctcacagtaa gaaagcactg atgcgctacg
     6661 aagacgttta catgcctgag gtgtacaaag cgattaacat tgcgcaaaac accgcatgga
     6721 aaatcaacaa gaaagtccta gcggtcgcca acgtaatcac caagtggaag cattgtccgg
     6781 tcgaggacat ccctgcgatt gagcgtgaag aactcccgat gaaaccggaa gacatcgaca
     6841 tgaatcctga ggctctcacc gcgtggaaac gtgctgccgc tgctgtgtac cgcaaggaca
     6901 aggctcgcaa gtctcgccgt atcagccttg agttcatgct tgagcaagcc aataagtttg
     6961 ctaaccataa ggccatctgg ttcccttaca acatggactg gcgcggtcgt gtttacgctg
     7021 tgtcaatgtt caacccgcaa ggtaacgata tgaccaaagg actgcttacg ctggcgaaag
     7081 gtaaaccaat cggtaaggaa ggttactact ggctgaaaat ccacggtgca aactgtgcgg
     7141 gtgtcgataa ggttccgttc cctgagcgca tcaagttcat tgaggaaaac cacgagaaca
     7201 tcatggcttg cgctaagtct ccactggaga acacttggtg ggctgagcaa gattctccgt
     7261 tctgcttcct tgcgttctgc tttgagtacg ctggggtaca gcaccacggc ctgagctata
     7321 actgctccct tccgctggcg tttgacgggt cttgctctgg catccagcac ttctccgcga
     7381 tgctccgaga tgaggtaggt ggtcgcgcgg ttaacttgct tcctagtgaa accgttcagg
     7441 acatctacgg gattgttgct aagaaagtca acgagattct acaagcagac gcaatcaatg
     7501 ggaccgataa cgaagtagtt accgtgaccg atgagaacac tggtgaaatc tctgagaaag
     7561 tcaagctggg cactaaggca ctggctggtc aatggctggc ttacggtgtt actcgcagtg
     7621 tgactaagcg ttcagtcatg acgctggctt acgggtccaa agagttcggc ttccgtcaac
     7681 aagtgctgga agataccatt cagccagcta ttgattccgg caagggtctg atgttcactc
     7741 agccgaatca ggctgctgga tacatggcta agctgatttg ggaatctgtg agcgtgacgg
     7801 tggtagctgc ggttgaagca atgaactggc ttaagtctgc tgctaagctg ctggctgctg
     7861 aggtcaaaga taagaagact ggagagattc ttcgcaagcg ttgcgctgtg cattgggtaa
     7921 ctcctgatgg tttccctgtg tggcaggaat acaagaagcc tattcagacg cgcttgaacc
     7981 tgatgttcct cggtcagttc cgcttacagc ctaccattaa caccaacaaa gatagcgaga
     8041 ttgatgcaca caaacaggag tctggtatcg ctcctaactt tgtacacagc caagacggta
     8101 gccaccttcg taagactgta gtgtgggcac acgagaagta cggaatcgaa tcttttgcac
     8161 tgattcacga ctccttcggt accattccgg ctgacgctgc gaacctgttc aaagcagtgc
     8221 gcgaaactat ggttgacaca tatgagtctt gtgatgtact ggctgatttc tacgaccagt
     8281 tcgctgacca gttgcacgag tctcaattgg acaaaatgcc agcacttccg gctaaaggta
     8341 acttgaacct ccgtgacatc ttagagtcgg acttcgcgtt cgcgtaatgg agattttcaa
     8401 catgctccct caatcggttg aatgtcgccc ttttgtcttt ggcgctggta aaccatatga
     8461 attttctatt gattgtgaca aaataaactt attccgtggt gtctttgcgt ttcttttata
     8521 tgttgccacc tttatgtatg tattttctac gtttgctaac atactgcgta ataaggagtc
     8581 ttaatcatgc cagttctttt gggtattccg tagaaaaagg aagagtatga gggaagcggt
     8641 gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga
     8701 accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca
     8761 cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc
     8821 tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc
     8881 tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg
     8941 cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc
     9001 cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt
     9061 ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc
     9121 gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa
     9181 tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa
     9241 ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact
     9301 tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca
     9361 gttggaagaa tttgtccact acgtgaaagg c
//
