LOCUS       sequence-331951-        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-VAMP1a
FEATURES             Location/Qualifiers
     source          1..9391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             47..835
                     /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"
     primer_bind     complement(917..934)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1051..1596)
                     /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(1131..1150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2220..2239
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2220..2238
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2486..2503
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3514..4593
                     /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             4657..5637
                     /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             5920..6369
                     /codon_start=1
                     /product="lysozyme from bacteriophage T7"
                     /label=T7 lysozyme
                     /note="inhibitor of T7 RNA polymerase"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAVLRAHHEVAPKASPSFDLKRWWEKNELVTSDR"
ORIGIN
        1 ttaatcatgc cagttctttt gggtattccg tagaaaaagg aagagtatga gggaagcggt
       61 gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga
      121 accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca
      181 cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc
      241 tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc
      301 tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg
      361 cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc
      421 cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt
      481 ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc
      541 gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa
      601 tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa
      661 ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact
      721 tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca
      781 gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaaacgga
      841 tcgcttcatg tggcagtaga aaaaagactg caccggtgcg tcagcagaat atgtgataca
      901 ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag
      961 cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca
     1021 gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca
     1081 tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca
     1141 ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg
     1201 tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt
     1261 aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg
     1321 ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg
     1381 cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc
     1441 taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa
     1501 gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca
     1561 gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat taaggggtct
     1621 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1681 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     1741 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1801 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1861 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1921 ccagatttat cagcaataaa ccagccagcc gattcgagct cgcccgggga tcgaccagtt
     1981 ggtgattttg aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat
     2041 ctgatccttc aactcagcaa aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc
     2101 agcgtaatgc tctgccagtg ttacaaccaa ttaacaagag gacatccggt caaataaaac
     2161 gaaaggctca gtcgaaagac tgggcctttc gttttgctga ggagacttag ggaccctact
     2221 aatacgactc actataggga gaaagaagga gcgacattgc tccgtgtatt cactcgttgg
     2281 aatgaataca cagtgcagtg tttattctgt tatttatgcc aaaaataaag gccactatca
     2341 ggcagctttg ttgttctgtt taccaagttc aggaggtaac tcataagaaa gacctgcagg
     2401 tgcagtaaag gaaaaaaaaa tgaaaaaatt attattcgca attcctttag ttgttccttt
     2461 ctattctcac tccgctgaaa ctgttcatca ccatcaccat cacgctgaaa ctgttgaaag
     2521 ttgtttagca aaaccccata cagaaaattc atttactaac gtctggaaag acgacaaaac
     2581 tttagatcgt tacgctaact atgagggctg tctgtggaat gctacaggcg ttgtagtttg
     2641 tactggtgac gaaactcagt gttacggtac atgggttcct attgggcttg ctatccctga
     2701 aaatgagggt ggtggctctg agggtggcgg ttctgagggt ggcggttctg agggtggcgg
     2761 tactaaacct cctgagtacg gtgatacacc tattccgggc tatacttata tcaaccctct
     2821 cgacggcact tatccgcctg gtactgagca aaaccccgct aatcctaatc cttctcttga
     2881 ggagtctcag cctcttaata ctttcatgtt tcagaataat aggttccgaa ataggcaggg
     2941 ggcattaact gtttatacgg gcactgttac tcaaggcact gaccccgtta aaacttatta
     3001 ccagtacact cctgtatcat caaaagccat gtatgacgct tactggaacg gtaaattcag
     3061 agactgcgct ttccattctg gctttaatga ggatccattc gtttgtgaat atcaaggcca
     3121 atcgtctgac ctgcctcaac ctcctgtcaa tgctggcggc ggctctggtg gtggttctgg
     3181 tggcggctct gagggtggtg gctctgaggg tggcggttct gagggtggcg gctctgaggg
     3241 aggcggttcc ggtggtggct cttcccaaat ggctcaagtc ggtgacggtg ataattcacc
     3301 tttaatgaat aatttccgtc aatatttacc ttccctccct caatcggttg aatgtcgccc
     3361 ttttgtcttt ggcgctggta aaccttacga gttcagtatc gactgcgata agatcaacct
     3421 gttccgcggt gtctttgcgt ttcttttata tgttgccacc tttatgtatg tattttctac
     3481 gtttgctaac atactgcgta ataaggagtc ttaatgaaat ttggaaactt tttgcttaca
     3541 taccaacctc cccaattttc ccaaacagag gtaatgaaac gtttggttaa attaggtcgc
     3601 atctctgagg agtgtggttt tgataccgta tggttactgg agcatcattt cacggagttt
     3661 ggtttgcttg gtaaccctta tgtcgctgct gcatatttac ttggcgcgac taaaaaattg
     3721 aatgtaggaa ctgccgctat tgttcttccc acagcccatc cagtacgcca acttgaagat
     3781 gtgaatttat tggatcaaat gtcaaaagga cgatttcggt ttggtatttg ccgagggctt
     3841 tacaacaagg actttcgcgt attcggcaca gatatgaata acagtcgcgc cttagcggaa
     3901 tgctggtacg ggctgataaa gaatggcatg acagagggat atatggaagc tgataatgaa
     3961 catatcaagt tccataaggt aaaagtaaac cccgcggcgt atagcagagg tggcgcaccg
     4021 gtttatgtgg tggctgaatc agcttcgacg actgagtggg ctgctcaatt tggcctaccg
     4081 atgatattaa gttggattat aaatactaac gaaaagaaag cacaacttga gctttataat
     4141 gaagtggctc aagaatatgg gcacgatatt cataatatcg accattgctt atcatatata
     4201 acatctgtag atcatgactc aattaaagcg aaagagattt gccggaaatt tctggggcat
     4261 tggtatgatt cttatgtgaa tgctacgact atttttgatg attcagacca aacaagaggt
     4321 tatgatttca ataaagggca gtggcgtgac tttgtattaa aaggacataa agatactaat
     4381 cgccgtattg attacagtta cgaaatcaat cccgtgggaa cgccgcagga atgtattgac
     4441 ataattcaaa aagacattga tgctacagga atatcaaata tttgttgtgg atttgaagct
     4501 aatggaacag tagacgaaat tattgcttcc atgaagctct tccagtctga tgtcatgcca
     4561 tttcttaaag aaaaacaacg ttcgctatta tattatggcg gtggcggtag cggcggtggc
     4621 ggtagcggcg gtggcggtag cggcggtggc ggtagcaaat ttggattgtt cttccttaac
     4681 ttcatcaatt caacaactgt tcaagaacag agtatagttc gcatgcagga aataacggag
     4741 tatgttgata agttgaattt tgaacagatt ttagtgtatg aaaatcattt ttcagataat
     4801 ggtgttgtcg gcgctcctct gactgtttct ggttttctgc tcggtttaac agagaaaatt
     4861 aaaattggtt cattaaatca catcattaca actcatcatc ctgtccgcat agcggaggaa
     4921 gcttgcttat tggatcagtt aagtgaaggg agatttattt tagggtttag tgattgcgaa
     4981 aaaaaagatg aaatgcattt ttttaatcgc ccggttgaat atcaacagca actatttgaa
     5041 gagtgttatg aaatcattaa cgatgcttta acaacaggct attgtaatcc agataacgat
     5101 ttttatagct tccctaaaat atctgtaaat ccccatgctt atacgccagg cggacctcgg
     5161 aaatatgtaa cagcaaccag tcatcatatt gttgagtggg cggccaaaaa aggtattcct
     5221 ctcatcttta agtgggatga ttctaatgat gttagatatg aatatgctga aagatataaa
     5281 gccgttgcgg ataaatatga cgttgaccta tcagagatag accatcagtt aatgatatta
     5341 gttaactata acgaagatag taataaagct aaacaagaga cgcgtgcatt tattagtgat
     5401 tatgttcttg aaatgcaccc taatgaaaat ttcgaaaata aacttgaaga aataattgca
     5461 gaaaacgctg tcggaaatta tacggagtgt ataactgcgg ctaagttggc aattgaaaag
     5521 tgtggtgcga aaagtgtatt gctgtccttt gaaccaatga atgatttgat gagccaaaaa
     5581 aatgtaatca atattgttga tgataatatt aagaagtacc acacggaata tacctaaact
     5641 taattaacgg cactcctcag caaatataat gaccctcttg ataacccaag agggcatttt
     5701 ttaatgccca tggcgtcaat tccgatttcc ggtattctag agcacagcta acaccacgtc
     5761 gtccctatct gctgccctag gtctatgagt ggttgctgga taactttacg ggcatgcata
     5821 aggctcgtat aatatattca gggagaccac aacggtttcc ctctacaaat aattttgttt
     5881 aacttttact agagttcctt cctcgaaagg aaaaaaaaaa tggctcgtgt acagtttaaa
     5941 caacgtgaat ctactgacgc aatctttgtt cactgctcgg ctaccaagcc aagtcagaat
     6001 gttggtgtcc gtgagattcg ccagtggcac aaagagcagg gttggctcga tgtgggatac
     6061 cactttatca tcaagcgaga cggtactgtg gaggcaggac gagatgagat ggctgtaggc
     6121 tctcacgcta agggttacaa ccacaactct atcggcgtct gccttgttgg tggtatcgac
     6181 gataaaggta agttcgacgc taactttacg ccagcccaaa tgcaatccct tcgctcactg
     6241 cttgtcacac tgctggctaa gtacgaaggc gctgtgcttc gcgcccatca tgaggtggcg
     6301 ccgaaggctt ccccttcgtt cgaccttaag cgttggtggg agaagaacga actggtcaca
     6361 tctgaccgta cctctaaccg tcgtctgcag cagacccagg ctcaggttga agaagttgtt
     6421 gacatcatcc gtgttaacgt tgacaaagtt ctggaacgtg accagaaact gtctgaactg
     6481 gacgaccgtg ctgacgctct gcaggctggt gcttctcagt tcgaatcttc tgctgctaaa
     6541 ctgaaacgta acacgattaa catcgctaag aacgacttct ctgacatcga actggctgct
     6601 atcccgttca acactctggc tgaccattac ggtgagcgtt tagctcgcga acagttggcc
     6661 cttgagcatg agtcttacga gatgggtgaa gcacgcttcc gcaagatgtt tgagcgtcaa
     6721 cttaaagctg gtgaggttgc ggataacgct gccgctaagc ctctcatcac taccctactc
     6781 cctaagatga ttgcacgcat caacgactgg tttgaggaag tgaaagctaa gcgcggcaag
     6841 cgcccgacag ccttccagtt cctgcaagaa atcaagccgg aagccgtagc gtacatcacc
     6901 attaagacca ctctggcttg cctaaccagt gctgacaata caaccgttca ggctgtagca
     6961 agcgcaatcg gtcgggccat tgaggacgag gctcgcttcg gtcgtatccg tgaccttgaa
     7021 gctaagcact tcaagaaaaa cgttgaggaa caactcaaca agcgcgtagg gcacgtctac
     7081 aagaaagcat ttatgcaagt tgtcgaggct gacatgctct ctaagggtct actcggtggc
     7141 gaggcgtggt cttcgtggca taaggaagac tctattcatg taggagtacg ctgcatcgag
     7201 atgctcattg agtcaaccgg aatggttagc ttacaccgcc aaaatgctgg cgtagtaggt
     7261 caagactctg agactatcga actcgcacct gaatacgctg aggctatcgc aacccgtgca
     7321 ggtgcgctgg ctggcatctc tccgatgttc caaccttgcg tagttcctcc taagccgtgg
     7381 actggcatta ctggtggtgg ctattgggct aacggtcgtc gtcctctggc gctggtgcgt
     7441 actcacagta agaaagcact gatgcgctac gaagacgttt acatgcctga ggtgtacaaa
     7501 gcgattaaca ttgcgcaaaa caccgcatgg aaaatcaaca agaaagtcct agcggtcgcc
     7561 aacgtaatca ccaagtggaa gcattgtccg gtcgaggaca tccctgcgat tgagcgtgaa
     7621 gaactcccga tgaaaccgga agacatcgac atgaatcctg aggctctcac cgcgtggaaa
     7681 cgtgctgccg ctgctgtgta ccgcaaggac aaggctcgca agtctcgccg tatcagcctt
     7741 gagttcatgc ttgagcaagc caataagttt gctaaccata aggccatctg gttcccttac
     7801 aacatggact ggcgcggtcg tgtttacgct gtgtcaatgt tcaacccgca aggtaacgat
     7861 atgaccaaag gactgcttac gctggcgaaa ggtaaaccaa tcggtaagga aggttactac
     7921 tggctgaaaa tccacggtgc aaactgtgcg ggtgtcgata aggttccgtt ccctgagcgc
     7981 atcaagttca ttgaggaaaa ccacgagaac atcatggctt gcgctaagtc tccactggag
     8041 aacacttggt gggctgagca agattctccg ttctgcttcc ttgcgttctg ctttgagtac
     8101 gctggggtac agcaccacgg cctgagctat aactgctccc ttccgctggc gtttgacggg
     8161 tcttgctctg gcatccagca cttctccgcg atgctccgag atgaggtagg tggtcgcgcg
     8221 gttaacttgc ttcctagtga aaccgttcag gacatctacg ggattgttgc taagaaagtc
     8281 aacgagattc tacaagcaga cgcaatcaat gggaccgata acgaagtagt taccgtgacc
     8341 gatgagaaca ctggtgaaat ctctgagaaa gtcaagctgg gcactaaggc actggctggt
     8401 caatggctgg cttacggtgt tactcgcagt gtgactaagc gttcagtcat gacgctggct
     8461 tacgggtcca aagagttcgg cttccgtcaa caagtgctgg aagataccat tcagccagct
     8521 attgattccg gcaagggtct gatgttcact cagccgaatc aggctgctgg atacatggct
     8581 aagctgattt gggaatctgt gagcgtgacg gtggtagctg cggttgaagc aatgaactgg
     8641 cttaagtctg ctgctaagct gctggctgct gaggtcaaag ataagaagac tggagagatt
     8701 cttcgcaagc gttgcgctgt gcattgggta actcctgatg gtttccctgt gtggcaggaa
     8761 tacaagaagc ctattcagac gcgcttgaac ctgatgttcc tcggtcagtt ccgcttacag
     8821 cctaccatta acaccaacaa agatagcgag attgatgcac acaaacagga gtctggtatc
     8881 gctcctaact ttgtacacag ccaagacggt agccaccttc gtaagactgt agtgtgggca
     8941 cacgagaagt acggaatcga atcttttgca ctgattcacg actccttcgg taccattccg
     9001 gctgacgctg cgaacctgtt caaagcagtg cgcgaaacta tggttgacac atatgagtct
     9061 tgtgatgtac tggctgattt ctacgaccag ttcgctgacc agttgcacga gtctcaattg
     9121 gacaaaatgc cagcacttcc ggctaaaggt aacttgaacc tccgtgacat cttagagtcg
     9181 gacttcgcgt tcgcgtaatg gagattttca acatgctccc tcaatcggtt gaatgtcgcc
     9241 cttttgtctt tggcgctggt aaaccatatg aattttctat tgattgtgac aaaataaact
     9301 tattccgtgg tgtctttgcg tttcttttat atgttgccac ctttatgtat gtattttcta
     9361 cgtttgctaa catactgcgt aataaggagt c
//
