LOCUS       sequence-331971-        9335 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 9335)
  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 9335)
  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-SNAP25c
FEATURES             Location/Qualifiers
     source          1..9335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(146..163)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(280..825)
                     /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(360..379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1449..1468
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1449..1467
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1716..1733
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2744..3823
                     /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             3887..4867
                     /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             5150..5602
                     /codon_start=1
                     /product="lysozyme from bacteriophage T7"
                     /label=T7 lysozyme
                     /note="inhibitor of T7 RNA polymerase"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAVLRAHHEVAPKASPSFDLKRWWEKNELVTSDRG"
     CDS             join(8611..9335,1..64)
                     /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 cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg
       61 caaaacggat cgcttcatgt ggcagtagaa aaaagactgc accggtgcgt cagcagaata
      121 tgtgatacag gatatattcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac
      181 tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat gccaggaaga
      241 tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc tccgcccccc
      301 tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga caggactata
      361 aagataccag gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg
      421 gtttaccggt gtcattccgc tgttatggcc gcgtttgtct cattccacgc ctgacactca
      481 gttccgggta ggcagttcgc tccaagctgg actgtatgca cgaacccccc gttcagtccg
      541 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggaaaga catgcaaaag
      601 caccactggc agcagccact ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc
      661 ggttaaggct aaactgaaag gacaagtttt ggtgactgcg ctcctccaag ccagttacct
      721 cggttcaaag agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt
      781 tcgttttcag agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt
      841 aaggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
      901 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
      961 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     1021 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     1081 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     1141 tcaccggctc cagatttatc agcaataaac cagccagccg attcgagctc gcccggggat
     1201 cgaccagttg gtgattttga acttttgctt tgccacggaa cggtctgcgt tgtcgggaag
     1261 atgcgtgatc tgatccttca actcagcaaa agttcgattt attcaacaaa gccgccgtcc
     1321 cgtcaagtca gcgtaatgct ctgccagtgt tacaaccaat taacaagagg acatccggtc
     1381 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttgctgag gagacttagg
     1441 gaccctacta atacgactca ctatagggag aaagaaggag cgacattgct ccgtgtattc
     1501 actcgttgga atgaatacac agtgcagtgt ttattctgtt atttatgcca aaaataaagg
     1561 ccactatcag gcagctttgt tgttctgttt accaagttca ggaggtaact cataagaaag
     1621 acctgcaggt gcagtaaaaa aaaaggaaaa tgaaaaaatt attattcgca attcctttag
     1681 ttgttccttt ctattctcac tccgctgaaa ctgttcatca ccatcaccat cacgctgaaa
     1741 ctgttgaaag ttgtttagca aaaccccata cagaaaattc atttactaac gtctggaaag
     1801 acgacaaaac tttagatcgt tacgctaact atgagggctg tctgtggaat gctacaggcg
     1861 ttgtagtttg tactggtgac gaaactcagt gttacggtac atgggttcct attgggcttg
     1921 ctatccctga aaatgagggt ggtggctctg agggtggcgg ttctgagggt ggcggttctg
     1981 agggtggcgg tactaaacct cctgagtacg gtgatacacc tattccgggc tatacttata
     2041 tcaaccctct cgacggcact tatccgcctg gtactgagca aaaccccgct aatcctaatc
     2101 cttctcttga ggagtctcag cctcttaata ctttcatgtt tcagaataat aggttccgaa
     2161 ataggcaggg ggcattaact gtttatacgg gcactgttac tcaaggcact gaccccgtta
     2221 aaacttatta ccagtacact cctgtatcat caaaagccat gtatgacgct tactggaacg
     2281 gtaaattcag agactgcgct ttccattctg gctttaatga ggatccattc gtttgtgaat
     2341 atcaaggcca atcgtctgac ctgcctcaac ctcctgtcaa tgctggcggc ggctctggtg
     2401 gtggttctgg tggcggctct gagggtggtg gctctgaggg tggcggttct gagggtggcg
     2461 gctctgaggg aggcggttcc ggtggtggct cttcccaaat ggctcaagtc ggtgacggtg
     2521 ataattcacc tttaatgaat aatttccgtc aatatttacc ttccctccct caatcggttg
     2581 aatgtcgccc ttttgtcttt ggcgctggta aaccttacga gttcagtatc gactgcgata
     2641 agatcaacct gttccgcggt gtctttgcgt ttcttttata tgttgccacc tttatgtatg
     2701 tattttctac gtttgctaac atactgcgta ataaggagtc ttaatgaaat ttggaaactt
     2761 tttgcttaca taccaacctc cccaattttc ccaaacagag gtaatgaaac gtttggttaa
     2821 attaggtcgc atctctgagg agtgtggttt tgataccgta tggttactgg agcatcattt
     2881 cacggagttt ggtttgcttg gtaaccctta tgtcgctgct gcatatttac ttggcgcgac
     2941 taaaaaattg aatgtaggaa ctgccgctat tgttcttccc acagcccatc cagtacgcca
     3001 acttgaagat gtgaatttat tggatcaaat gtcaaaagga cgatttcggt ttggtatttg
     3061 ccgagggctt tacaacaagg actttcgcgt attcggcaca gatatgaata acagtcgcgc
     3121 cttagcggaa tgctggtacg ggctgataaa gaatggcatg acagagggat atatggaagc
     3181 tgataatgaa catatcaagt tccataaggt aaaagtaaac cccgcggcgt atagcagagg
     3241 tggcgcaccg gtttatgtgg tggctgaatc agcttcgacg actgagtggg ctgctcaatt
     3301 tggcctaccg atgatattaa gttggattat aaatactaac gaaaagaaag cacaacttga
     3361 gctttataat gaagtggctc aagaatatgg gcacgatatt cataatatcg accattgctt
     3421 atcatatata acatctgtag atcatgactc aattaaagcg aaagagattt gccggaaatt
     3481 tctggggcat tggtatgatt cttatgtgaa tgctacgact atttttgatg attcagacca
     3541 aacaagaggt tatgatttca ataaagggca gtggcgtgac tttgtattaa aaggacataa
     3601 agatactaat cgccgtattg attacagtta cgaaatcaat cccgtgggaa cgccgcagga
     3661 atgtattgac ataattcaaa aagacattga tgctacagga atatcaaata tttgttgtgg
     3721 atttgaagct aatggaacag tagacgaaat tattgcttcc atgaagctct tccagtctga
     3781 tgtcatgcca tttcttaaag aaaaacaacg ttcgctatta tattatggcg gtggcggtag
     3841 cggcggtggc ggtagcggcg gtggcggtag cggcggtggc ggtagcaaat ttggattgtt
     3901 cttccttaac ttcatcaatt caacaactgt tcaagaacag agtatagttc gcatgcagga
     3961 aataacggag tatgttgata agttgaattt tgaacagatt ttagtgtatg aaaatcattt
     4021 ttcagataat ggtgttgtcg gcgctcctct gactgtttct ggttttctgc tcggtttaac
     4081 agagaaaatt aaaattggtt cattaaatca catcattaca actcatcatc ctgtccgcat
     4141 agcggaggaa gcttgcttat tggatcagtt aagtgaaggg agatttattt tagggtttag
     4201 tgattgcgaa aaaaaagatg aaatgcattt ttttaatcgc ccggttgaat atcaacagca
     4261 actatttgaa gagtgttatg aaatcattaa cgatgcttta acaacaggct attgtaatcc
     4321 agataacgat ttttatagct tccctaaaat atctgtaaat ccccatgctt atacgccagg
     4381 cggacctcgg aaatatgtaa cagcaaccag tcatcatatt gttgagtggg cggccaaaaa
     4441 aggtattcct ctcatcttta agtgggatga ttctaatgat gttagatatg aatatgctga
     4501 aagatataaa gccgttgcgg ataaatatga cgttgaccta tcagagatag accatcagtt
     4561 aatgatatta gttaactata acgaagatag taataaagct aaacaagaga cgcgtgcatt
     4621 tattagtgat tatgttcttg aaatgcaccc taatgaaaat ttcgaaaata aacttgaaga
     4681 aataattgca gaaaacgctg tcggaaatta tacggagtgt ataactgcgg ctaagttggc
     4741 aattgaaaag tgtggtgcga aaagtgtatt gctgtccttt gaaccaatga atgatttgat
     4801 gagccaaaaa aatgtaatca atattgttga tgataatatt aagaagtacc acacggaata
     4861 tacctaaact taattaacgg cactcctcag caaatataat gaccctcttg ataacccaag
     4921 agggcatttt ttaatgccca tggcgtcaat tccgatttcc ggtattctag agcacagcta
     4981 acaccacgtc gtccctatct gctgccctag gtctatgagt ggttgctgga taactttacg
     5041 ggcatgcata aggctcgtat aatatattca gggagaccac aacggtttcc ctctacaaat
     5101 aattttgttt aacttttact agagttcctt cctcgaaagg aaaaaaaaaa tggctcgtgt
     5161 acagtttaaa caacgtgaat ctactgacgc aatctttgtt cactgctcgg ctaccaagcc
     5221 aagtcagaat gttggtgtcc gtgagattcg ccagtggcac aaagagcagg gttggctcga
     5281 tgtgggatac cactttatca tcaagcgaga cggtactgtg gaggcaggac gagatgagat
     5341 ggctgtaggc tctcacgcta agggttacaa ccacaactct atcggcgtct gccttgttgg
     5401 tggtatcgac gataaaggta agttcgacgc taactttacg ccagcccaaa tgcaatccct
     5461 tcgctcactg cttgtcacac tgctggctaa gtacgaaggc gctgtgcttc gcgcccatca
     5521 tgaggtggcg ccgaaggctt ccccttcgtt cgaccttaag cgttggtggg agaagaacga
     5581 actggtcact tctgaccgtg gtagcggcgg tggtgcgagt ggtggtgcgg acatgggtaa
     5641 cgaaatcgac acccagaacc gtcagatcga ccgtatcatg gaaaaagctg acggcggtag
     5701 cgcaggcagt ggagcgggcg gtaacacgat taacatcgct aagaacgact tctctgacat
     5761 cgaactggct gctatcccgt tcaacactct ggctgaccat tacggtgagc gtttagctcg
     5821 cgaacagttg gcccttgagc atgagtctta cgagatgggt gaagcacgct tccgcaagat
     5881 gtttgagcgt caacttaaag ctggtgaggt tgcggataac gctgccgcta agcctctcat
     5941 cactacccta ctccctaaga tgattgcacg catcaacgac tggtttgagg aagtgaaagc
     6001 taagcgcggc aagcgcccga cagccttcca gttcctgcaa gaaatcaagc cggaagccgt
     6061 agcgtacatc accattaaga ccactctggc ttgcctaacc agtgctgaca atacaaccgt
     6121 tcaggctgta gcaagcgcaa tcggtcgggc cattgaggac gaggctcgct tcggtcgtat
     6181 ccgtgacctt gaagctaagc acttcaagaa aaacgttgag gaacaactca acaagcgcgt
     6241 agggcacgtc tacaagaaag catttatgca agttgtcgag gctgacatgc tctctaaggg
     6301 tctactcggt ggcgaggcgt ggtcttcgtg gcataaggaa gactctattc atgtaggagt
     6361 acgctgcatc gagatgctca ttgagtcaac cggaatggtt agcttacacc gccaaaatgc
     6421 tggcgtagta ggtcaagact ctgagactat cgaactcgca cctgaatacg ctgaggctat
     6481 cgcaacccgt gcaggtgcgc tggctggcat ctctccgatg ttccaacctt gcgtagttcc
     6541 tcctaagccg tggactggca ttactggtgg tggctattgg gctaacggtc gtcgtcctct
     6601 ggcgctggtg cgtactcaca gtaagaaagc actgatgcgc tacgaagacg tttacatgcc
     6661 tgaggtgtac aaagcgatta acattgcgca aaacaccgca tggaaaatca acaagaaagt
     6721 cctagcggtc gccaacgtaa tcaccaagtg gaagcattgt ccggtcgagg acatccctgc
     6781 gattgagcgt gaagaactcc cgatgaaacc ggaagacatc gacatgaatc ctgaggctct
     6841 caccgcgtgg aaacgtgctg ccgctgctgt gtaccgcaag gacaaggctc gcaagtctcg
     6901 ccgtatcagc cttgagttca tgcttgagca agccaataag tttgctaacc ataaggccat
     6961 ctggttccct tacaacatgg actggcgcgg tcgtgtttac gctgtgtcaa tgttcaaccc
     7021 gcaaggtaac gatatgacca aaggactgct tacgctggcg aaaggtaaac caatcggtaa
     7081 ggaaggttac tactggctga aaatccacgg tgcaaactgt gcgggtgtcg ataaggttcc
     7141 gttccctgag cgcatcaagt tcattgagga aaaccacgag aacatcatgg cttgcgctaa
     7201 gtctccactg gagaacactt ggtgggctga gcaagattct ccgttctgct tccttgcgtt
     7261 ctgctttgag tacgctgggg tacagcacca cggcctgagc tataactgct cccttccgct
     7321 ggcgtttgac gggtcttgct ctggcatcca gcacttctcc gcgatgctcc gagatgaggt
     7381 aggtggtcgc gcggttaact tgcttcctag tgaaaccgtt caggacatct acgggattgt
     7441 tgctaagaaa gtcaacgaga ttctacaagc agacgcaatc aatgggaccg ataacgaagt
     7501 agttaccgtg accgatgaga acactggtga aatctctgag aaagtcaagc tgggcactaa
     7561 ggcactggct ggtcaatggc tggcttacgg tgttactcgc agtgtgacta agcgttcagt
     7621 catgacgctg gcttacgggt ccaaagagtt cggcttccgt caacaagtgc tggaagatac
     7681 cattcagcca gctattgatt ccggcaaggg tctgatgttc actcagccga atcaggctgc
     7741 tggatacatg gctaagctga tttgggaatc tgtgagcgtg acggtggtag ctgcggttga
     7801 agcaatgaac tggcttaagt ctgctgctaa gctgctggct gctgaggtca aagataagaa
     7861 gactggagag attcttcgca agcgttgcgc tgttcattgg gtaactcctg atggtttccc
     7921 tgtgtggcag gaatacaaga agcctattca gacgcgcttg aacctgatgt tcctcggtca
     7981 gttccgctta cagcctacca ttaacaccaa caaagatagc gagattgatg cacacaaaca
     8041 ggagtctggt atcgctccta actttgtaca cagccaagac ggtagccacc ttcgtaagac
     8101 tgtagtgtgg gcacacgaga agtacggaat cgaatctttt gcactgattc acgactcctt
     8161 cggtaccatt ccggctgacg ctgcgaacct gttcaaagca gtgcgcgaaa ctatggttga
     8221 cacatatgag tcttgtgatg tactggctga tttctacgac cagttcgctg accagttgca
     8281 cgagtctcaa ttggacaaaa tgccagcact tccggctaaa ggtaacttga acctccgtga
     8341 catcttagag tcggacttcg cgttcgcgta atggagattt tcaacatgct ccctcaatcg
     8401 gttgaatgtc gcccttttgt ctttggcgct ggtaaaccat atgaattttc tattgattgt
     8461 gacaaaataa acttattccg tggtgtcttt gcgtttcttt tatatgttgc cacctttatg
     8521 tatgtatttt ctacgtttgc taacatactg cgtaataagg agtcttaatc atgccagttc
     8581 ttttgggtat tccgtagaaa aaggaagagt atgagggaag cggtgatcgc cgaagtatcg
     8641 actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc
     8701 gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg
     8761 ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt
     8821 ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt
     8881 gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga
     8941 gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat
     9001 ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg
     9061 gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta
     9121 acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg
     9181 tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac
     9241 tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct
     9301 tatcttggac aagaagaaga tcgcttggcc tcgcg
//
