LOCUS       sequence-331970-        9333 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 9333)
  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 9333)
  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-SNAP25b
FEATURES             Location/Qualifiers
     source          1..9333
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(88..105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(222..767)
                     /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(302..321)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1391..1410
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1391..1409
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1656..1673
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2684..3763
                     /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             3827..4807
                     /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             5090..5542
                     /codon_start=1
                     /product="lysozyme from bacteriophage T7"
                     /label=T7 lysozyme
                     /note="inhibitor of T7 RNA polymerase"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAVLRAHHEVAPKASPSFDLKRWWEKNELVTSDRG"
     CDS             join(8551..9333,1..6)
                     /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 ggcaaaacgg atcgcttcat gtggcagtag aaaaaagact gcaccggtgc gtcagcagaa
       61 tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg
      121 actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa
      181 gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc
      241 cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta
      301 taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt tcctgccttt
      361 cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac gcctgacact
      421 cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc ccgttcagtc
      481 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa
      541 agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg aagtcatgcg
      601 ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca agccagttac
      661 ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc aaggcggttt
      721 tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa gatcatctta
      781 ttaaggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
      841 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
      901 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
      961 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     1021 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     1081 gctcaccggc tccagattta tcagcaataa accagccagc cgattcgagc tcgcccgggg
     1141 atcgaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga
     1201 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt
     1261 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaacaaga ggacatccgg
     1321 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttgctg aggagactta
     1381 gggaccctac taatacgact cactataggg agaaagaagg agcgacattg ctccgtgtat
     1441 tcactcgttg gaatgaatac acagtgcagt gtttattctg ttatttatgc caaaaataaa
     1501 ggccactatc aggcagcttt gttgttctgt ttaccaagtt caggaggtaa ctcataagaa
     1561 agacctgcag gtgcagtaaa aaggaaaaaa tgaaaaaatt attattcgca attcctttag
     1621 ttgttccttt ctattctcac tccgctgaaa ctgttcatca ccatcaccat cacgctgaaa
     1681 ctgttgaaag ttgtttagca aaaccccata cagaaaattc atttactaac gtctggaaag
     1741 acgacaaaac tttagatcgt tacgctaact atgagggctg tctgtggaat gctacaggcg
     1801 ttgtagtttg tactggtgac gaaactcagt gttacggtac atgggttcct attgggcttg
     1861 ctatccctga aaatgagggt ggtggctctg agggtggcgg ttctgagggt ggcggttctg
     1921 agggtggcgg tactaaacct cctgagtacg gtgatacacc tattccgggc tatacttata
     1981 tcaaccctct cgacggcact tatccgcctg gtactgagca aaaccccgct aatcctaatc
     2041 cttctcttga ggagtctcag cctcttaata ctttcatgtt tcagaataat aggttccgaa
     2101 ataggcaggg ggcattaact gtttatacgg gcactgttac tcaaggcact gaccccgtta
     2161 aaacttatta ccagtacact cctgtatcat caaaagccat gtatgacgct tactggaacg
     2221 gtaaattcag agactgcgct ttccattctg gctttaatga ggatccattc gtttgtgaat
     2281 atcaaggcca atcgtctgac ctgcctcaac ctcctgtcaa tgctggcggc ggctctggtg
     2341 gtggttctgg tggcggctct gagggtggtg gctctgaggg tggcggttct gagggtggcg
     2401 gctctgaggg aggcggttcc ggtggtggct cttcccaaat ggctcaagtc ggtgacggtg
     2461 ataattcacc tttaatgaat aatttccgtc aatatttacc ttccctccct caatcggttg
     2521 aatgtcgccc ttttgtcttt ggcgctggta aaccttacga gttcagtatc gactgcgata
     2581 agatcaacct gttccgcggt gtctttgcgt ttcttttata tgttgccacc tttatgtatg
     2641 tattttctac gtttgctaac atactgcgta ataaggagtc ttaatgaaat ttggaaactt
     2701 tttgcttaca taccaacctc cccaattttc ccaaacagag gtaatgaaac gtttggttaa
     2761 attaggtcgc atctctgagg agtgtggttt tgataccgta tggttactgg agcatcattt
     2821 cacggagttt ggtttgcttg gtaaccctta tgtcgctgct gcatatttac ttggcgcgac
     2881 taaaaaattg aatgtaggaa ctgccgctat tgttcttccc acagcccatc cagtacgcca
     2941 acttgaagat gtgaatttat tggatcaaat gtcaaaagga cgatttcggt ttggtatttg
     3001 ccgagggctt tacaacaagg actttcgcgt attcggcaca gatatgaata acagtcgcgc
     3061 cttagcggaa tgctggtacg ggctgataaa gaatggcatg acagagggat atatggaagc
     3121 tgataatgaa catatcaagt tccataaggt aaaagtaaac cccgcggcgt atagcagagg
     3181 tggcgcaccg gtttatgtgg tggctgaatc agcttcgacg actgagtggg ctgctcaatt
     3241 tggcctaccg atgatattaa gttggattat aaatactaac gaaaagaaag cacaacttga
     3301 gctttataat gaagtggctc aagaatatgg gcacgatatt cataatatcg accattgctt
     3361 atcatatata acatctgtag atcatgactc aattaaagcg aaagagattt gccggaaatt
     3421 tctggggcat tggtatgatt cttatgtgaa tgctacgact atttttgatg attcagacca
     3481 aacaagaggt tatgatttca ataaagggca gtggcgtgac tttgtattaa aaggacataa
     3541 agatactaat cgccgtattg attacagtta cgaaatcaat cccgtgggaa cgccgcagga
     3601 atgtattgac ataattcaaa aagacattga tgctacagga atatcaaata tttgttgtgg
     3661 atttgaagct aatggaacag tagacgaaat tattgcttcc atgaagctct tccagtctga
     3721 tgtcatgcca tttcttaaag aaaaacaacg ttcgctatta tattatggcg gtggcggtag
     3781 cggcggtggc ggtagcggcg gtggcggtag cggcggtggc ggtagcaaat ttggattgtt
     3841 cttccttaac ttcatcaatt caacaactgt tcaagaacag agtatagttc gcatgcagga
     3901 aataacggag tatgttgata agttgaattt tgaacagatt ttagtgtatg aaaatcattt
     3961 ttcagataat ggtgttgtcg gcgctcctct gactgtttct ggttttctgc tcggtttaac
     4021 agagaaaatt aaaattggtt cattaaatca catcattaca actcatcatc ctgtccgcat
     4081 agcggaggaa gcttgcttat tggatcagtt aagtgaaggg agatttattt tagggtttag
     4141 tgattgcgaa aaaaaagatg aaatgcattt ttttaatcgc ccggttgaat atcaacagca
     4201 actatttgaa gagtgttatg aaatcattaa cgatgcttta acaacaggct attgtaatcc
     4261 agataacgat ttttatagct tccctaaaat atctgtaaat ccccatgctt atacgccagg
     4321 cggacctcgg aaatatgtaa cagcaaccag tcatcatatt gttgagtggg cggccaaaaa
     4381 aggtattcct ctcatcttta agtgggatga ttctaatgat gttagatatg aatatgctga
     4441 aagatataaa gccgttgcgg ataaatatga cgttgaccta tcagagatag accatcagtt
     4501 aatgatatta gttaactata acgaagatag taataaagct aaacaagaga cgcgtgcatt
     4561 tattagtgat tatgttcttg aaatgcaccc taatgaaaat ttcgaaaata aacttgaaga
     4621 aataattgca gaaaacgctg tcggaaatta tacggagtgt ataactgcgg ctaagttggc
     4681 aattgaaaag tgtggtgcga aaagtgtatt gctgtccttt gaaccaatga atgatttgat
     4741 gagccaaaaa aatgtaatca atattgttga tgataatatt aagaagtacc acacggaata
     4801 tacctaaact taattaacgg cactcctcag caaatataat gaccctcttg ataacccaag
     4861 agggcatttt ttaatgccca tggcgtcaat tccgatttcc ggtattctag agcacagcta
     4921 acaccacgtc gtccctatct gctgccctag gtctatgagt ggttgctgga taactttacg
     4981 ggcatgcata aggctcgtat aatatattca gggagaccac aacggtttcc ctctacaaat
     5041 aattttgttt aacttttact agagttcctt cctcgaaagg aaaaaaaaaa tggctcgtgt
     5101 acagtttaaa caacgtgaat ctactgacgc aatctttgtt cactgctcgg ctaccaagcc
     5161 aagtcagaat gttggtgtcc gtgagattcg ccagtggcac aaagagcagg gttggctcga
     5221 tgtgggatac cactttatca tcaagcgaga cggtactgtg gaggcaggac gagatgagat
     5281 ggctgtaggc tctcacgcta agggttacaa ccacaactct atcggcgtct gccttgttgg
     5341 tggtatcgac gataaaggta agttcgacgc taactttacg ccagcccaaa tgcaatccct
     5401 tcgctcactg cttgtcacac tgctggctaa gtacgaaggc gctgtgcttc gcgcccatca
     5461 tgaggtggcg ccgaaggctt ccccttcgtt cgaccttaag cgttggtggg agaagaacga
     5521 actggtcact tctgaccgtg gtagcggcgg tggtgcgagt ggtggtgcgg acatgggtaa
     5581 cgaaatcgac acccagaacc gtcagatcga ccgtatcatg gaaaaagctg acggcggtag
     5641 cgcaggcagt ggagcgggcg gtaacacgat taacatcgct aagaacgact tctctgacat
     5701 cgaactggct gctatcccgt tcaacactct ggctgaccat tacggtgagc gtttagctcg
     5761 cgaacagttg gcccttgagc atgagtctta cgagatgggt gaagcacgct tccgcaagat
     5821 gtttgagcgt caacttaaag ctggtgaggt tgcggataac gctgccgcta agcctctcat
     5881 cactacccta ctccctaaga tgattgcacg catcaacgac tggtttgagg aagtgaaagc
     5941 taagcgcggc aagcgcccga cagccttcca gttcctgcaa gaaatcaagc cggaagccgt
     6001 agcgtacatc accattaaga ccactctggc ttgcctaacc agtgctgaca atacaaccgt
     6061 tcaggctgta gcaagcgcaa tcggtcgggc cattgaggac gaggctcgct tcggtcgtat
     6121 ccgtgacctt gaagctaagc acttcaagaa aaacgttgag gaacaactca acaagcgcgt
     6181 agggcacgtc tacaagaaag catttatgca agttgtcgag gctgacatgc tctctaaggg
     6241 tctactcggt ggcgaggcgt ggtcttcgtg gcataaggaa gactctattc atgtaggagt
     6301 acgctgcatc gagatgctca ttgagtcaac cggaatggtt agcttacacc gccaaaatgc
     6361 tggcgtagta ggtcaagact ctgagactat cgaactcgca cctgaatacg ctgaggctat
     6421 cgcaacccgt gcaggtgcgc tggctggcat ctctccgatg ttccaacctt gcgtagttcc
     6481 tcctaagccg tggactggca ttactggtgg tggctattgg gctaacggtc gtcgtcctct
     6541 ggcgctggtg cgtactcaca gtaagaaagc actgatgcgc tacgaagacg tttacatgcc
     6601 tgaggtgtac aaagcgatta acattgcgca aaacaccgca tggaaaatca acaagaaagt
     6661 cctagcggtc gccaacgtaa tcaccaagtg gaagcattgt ccggtcgagg acatccctgc
     6721 gattgagcgt gaagaactcc cgatgaaacc ggaagacatc gacatgaatc ctgaggctct
     6781 caccgcgtgg aaacgtgctg ccgctgctgt gtaccgcaag gacaaggctc gcaagtctcg
     6841 ccgtatcagc cttgagttca tgcttgagca agccaataag tttgctaacc ataaggccat
     6901 ctggttccct tacaacatgg actggcgcgg tcgtgtttac gctgtgtcaa tgttcaaccc
     6961 gcaaggtaac gatatgacca aaggactgct tacgctggcg aaaggtaaac caatcggtaa
     7021 ggaaggttac tactggctga aaatccacgg tgcaaactgt gcgggtgtcg ataaggttcc
     7081 gttccctgag cgcatcaagt tcattgagga aaaccacgag aacatcatgg cttgcgctaa
     7141 gtctccactg gagaacactt ggtgggctga gcaagattct ccgttctgct tccttgcgtt
     7201 ctgctttgag tacgctgggg tacagcacca cggcctgagc tataactgct cccttccgct
     7261 ggcgtttgac gggtcttgct ctggcatcca gcacttctcc gcgatgctcc gagatgaggt
     7321 aggtggtcgc gcggttaact tgcttcctag tgaaaccgtt caggacatct acgggattgt
     7381 tgctaagaaa gtcaacgaga ttctacaagc agacgcaatc aatgggaccg ataacgaagt
     7441 agttaccgtg accgatgaga acactggtga aatctctgag aaagtcaagc tgggcactaa
     7501 ggcactggct ggtcaatggc tggcttacgg tgttactcgc agtgtgacta agcgttcagt
     7561 catgacgctg gcttacgggt ccaaagagtt cggcttccgt caacaagtgc tggaagatac
     7621 cattcagcca gctattgatt ccggcaaggg tctgatgttc actcagccga atcaggctgc
     7681 tggatacatg gctaagctga tttgggaatc tgtgagcgtg acggtggtag ctgcggttga
     7741 agcaatgaac tggcttaagt ctgctgctaa gctgctggct gctgaggtca aagataagaa
     7801 gactggagag attcttcgca agcgttgcgc tgttcattgg gtaactcctg atggtttccc
     7861 tgtgtggcag gaatacaaga agcctattca gacgcgcttg aacctgatgt tcctcggtca
     7921 gttccgctta cagcctacca ttaacaccaa caaagatagc gagattgatg cacacaaaca
     7981 ggagtctggt atcgctccta actttgtaca cagccaagac ggtagccacc ttcgtaagac
     8041 tgtagtgtgg gcacacgaga agtacggaat cgaatctttt gcactgattc acgactcctt
     8101 cggtaccatt ccggctgacg ctgcgaacct gttcaaagca gtgcgcgaaa ctatggttga
     8161 cacatatgag tcttgtgatg tactggctga tttctacgac cagttcgctg accagttgca
     8221 cgagtctcaa ttggacaaaa tgccagcact tccggctaaa ggtaacttga acctccgtga
     8281 catcttagag tcggacttcg cgttcgcgta atggagattt tcaacatgct ccctcaatcg
     8341 gttgaatgtc gcccttttgt ctttggcgct ggtaaaccat atgaattttc tattgattgt
     8401 gacaaaataa acttattccg tggtgtcttt gcgtttcttt tatatgttgc cacctttatg
     8461 tatgtatttt ctacgtttgc taacatactg cgtaataagg agtcttaatc atgccagttc
     8521 ttttgggtat tccgtagaaa aaggaagagt atgagggaag cggtgatcgc cgaagtatcg
     8581 actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc
     8641 gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg
     8701 ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt
     8761 ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt
     8821 gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga
     8881 gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat
     8941 ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg
     9001 gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta
     9061 acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg
     9121 tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac
     9181 tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct
     9241 tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc
     9301 cactacgtga aaggcgagat caccaaggta gtc
//
