LOCUS       sequence-331963-        9334 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 9334)
  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 9334)
  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-SNAP25a
FEATURES             Location/Qualifiers
     source          1..9334
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(148..165)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(282..827)
                     /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(362..381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1451..1470
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1451..1469
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1717..1734
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2745..3824
                     /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             3888..4868
                     /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             5151..5603
                     /codon_start=1
                     /product="lysozyme from bacteriophage T7"
                     /label=T7 lysozyme
                     /note="inhibitor of T7 RNA polymerase"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAVLRAHHEVAPKASPSFDLKRWWEKNELVTSDRG"
     CDS             join(8612..9334,1..66)
                     /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 cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc
       61 ggcaaaacgg atcgcttcat gtggcagtag aaaaaagact gcaccggtgc gtcagcagaa
      121 tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg
      181 actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa
      241 gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc
      301 cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta
      361 taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt tcctgccttt
      421 cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac gcctgacact
      481 cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc ccgttcagtc
      541 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa
      601 agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg aagtcatgcg
      661 ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca agccagttac
      721 ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc aaggcggttt
      781 tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa gatcatctta
      841 ttaaggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
      901 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
      961 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     1021 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     1081 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     1141 gctcaccggc tccagattta tcagcaataa accagccagc cgattcgagc tcgcccgggg
     1201 atcgaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga
     1261 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt
     1321 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaacaaga ggacatccgg
     1381 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttgctg aggagactta
     1441 gggaccctac taatacgact cactataggg agaaagaagg agcgacattg ctccgtgtat
     1501 tcactcgttg gaatgaatac acagtgcagt gtttattctg ttatttatgc caaaaataaa
     1561 ggccactatc aggcagcttt gttgttctgt ttaccaagtt caggaggtaa ctcataagaa
     1621 agacctgcag gtgcagtaaa ggaaaaaaaa atgaaaaaat tattattcgc aattccttta
     1681 gttgttcctt tctattctca ctccgctgaa actgttcatc accatcacca tcacgctgaa
     1741 actgttgaaa gttgtttagc aaaaccccat acagaaaatt catttactaa cgtctggaaa
     1801 gacgacaaaa ctttagatcg ttacgctaac tatgagggct gtctgtggaa tgctacaggc
     1861 gttgtagttt gtactggtga cgaaactcag tgttacggta catgggttcc tattgggctt
     1921 gctatccctg aaaatgaggg tggtggctct gagggtggcg gttctgaggg tggcggttct
     1981 gagggtggcg gtactaaacc tcctgagtac ggtgatacac ctattccggg ctatacttat
     2041 atcaaccctc tcgacggcac ttatccgcct ggtactgagc aaaaccccgc taatcctaat
     2101 ccttctcttg aggagtctca gcctcttaat actttcatgt ttcagaataa taggttccga
     2161 aataggcagg gggcattaac tgtttatacg ggcactgtta ctcaaggcac tgaccccgtt
     2221 aaaacttatt accagtacac tcctgtatca tcaaaagcca tgtatgacgc ttactggaac
     2281 ggtaaattca gagactgcgc tttccattct ggctttaatg aggatccatt cgtttgtgaa
     2341 tatcaaggcc aatcgtctga cctgcctcaa cctcctgtca atgctggcgg cggctctggt
     2401 ggtggttctg gtggcggctc tgagggtggt ggctctgagg gtggcggttc tgagggtggc
     2461 ggctctgagg gaggcggttc cggtggtggc tcttcccaaa tggctcaagt cggtgacggt
     2521 gataattcac ctttaatgaa taatttccgt caatatttac cttccctccc tcaatcggtt
     2581 gaatgtcgcc cttttgtctt tggcgctggt aaaccttacg agttcagtat cgactgcgat
     2641 aagatcaacc tgttccgcgg tgtctttgcg tttcttttat atgttgccac ctttatgtat
     2701 gtattttcta cgtttgctaa catactgcgt aataaggagt cttaatgaaa tttggaaact
     2761 ttttgcttac ataccaacct ccccaatttt cccaaacaga ggtaatgaaa cgtttggtta
     2821 aattaggtcg catctctgag gagtgtggtt ttgataccgt atggttactg gagcatcatt
     2881 tcacggagtt tggtttgctt ggtaaccctt atgtcgctgc tgcatattta cttggcgcga
     2941 ctaaaaaatt gaatgtagga actgccgcta ttgttcttcc cacagcccat ccagtacgcc
     3001 aacttgaaga tgtgaattta ttggatcaaa tgtcaaaagg acgatttcgg tttggtattt
     3061 gccgagggct ttacaacaag gactttcgcg tattcggcac agatatgaat aacagtcgcg
     3121 ccttagcgga atgctggtac gggctgataa agaatggcat gacagaggga tatatggaag
     3181 ctgataatga acatatcaag ttccataagg taaaagtaaa ccccgcggcg tatagcagag
     3241 gtggcgcacc ggtttatgtg gtggctgaat cagcttcgac gactgagtgg gctgctcaat
     3301 ttggcctacc gatgatatta agttggatta taaatactaa cgaaaagaaa gcacaacttg
     3361 agctttataa tgaagtggct caagaatatg ggcacgatat tcataatatc gaccattgct
     3421 tatcatatat aacatctgta gatcatgact caattaaagc gaaagagatt tgccggaaat
     3481 ttctggggca ttggtatgat tcttatgtga atgctacgac tatttttgat gattcagacc
     3541 aaacaagagg ttatgatttc aataaagggc agtggcgtga ctttgtatta aaaggacata
     3601 aagatactaa tcgccgtatt gattacagtt acgaaatcaa tcccgtggga acgccgcagg
     3661 aatgtattga cataattcaa aaagacattg atgctacagg aatatcaaat atttgttgtg
     3721 gatttgaagc taatggaaca gtagacgaaa ttattgcttc catgaagctc ttccagtctg
     3781 atgtcatgcc atttcttaaa gaaaaacaac gttcgctatt atattatggc ggtggcggta
     3841 gcggcggtgg cggtagcggc ggtggcggta gcggcggtgg cggtagcaaa tttggattgt
     3901 tcttccttaa cttcatcaat tcaacaactg ttcaagaaca gagtatagtt cgcatgcagg
     3961 aaataacgga gtatgttgat aagttgaatt ttgaacagat tttagtgtat gaaaatcatt
     4021 tttcagataa tggtgttgtc ggcgctcctc tgactgtttc tggttttctg ctcggtttaa
     4081 cagagaaaat taaaattggt tcattaaatc acatcattac aactcatcat cctgtccgca
     4141 tagcggagga agcttgctta ttggatcagt taagtgaagg gagatttatt ttagggttta
     4201 gtgattgcga aaaaaaagat gaaatgcatt tttttaatcg cccggttgaa tatcaacagc
     4261 aactatttga agagtgttat gaaatcatta acgatgcttt aacaacaggc tattgtaatc
     4321 cagataacga tttttatagc ttccctaaaa tatctgtaaa tccccatgct tatacgccag
     4381 gcggacctcg gaaatatgta acagcaacca gtcatcatat tgttgagtgg gcggccaaaa
     4441 aaggtattcc tctcatcttt aagtgggatg attctaatga tgttagatat gaatatgctg
     4501 aaagatataa agccgttgcg gataaatatg acgttgacct atcagagata gaccatcagt
     4561 taatgatatt agttaactat aacgaagata gtaataaagc taaacaagag acgcgtgcat
     4621 ttattagtga ttatgttctt gaaatgcacc ctaatgaaaa tttcgaaaat aaacttgaag
     4681 aaataattgc agaaaacgct gtcggaaatt atacggagtg tataactgcg gctaagttgg
     4741 caattgaaaa gtgtggtgcg aaaagtgtat tgctgtcctt tgaaccaatg aatgatttga
     4801 tgagccaaaa aaatgtaatc aatattgttg atgataatat taagaagtac cacacggaat
     4861 atacctaaac ttaattaacg gcactcctca gcaaatataa tgaccctctt gataacccaa
     4921 gagggcattt tttaatgccc atggcgtcaa ttccgatttc cggtattcta gagcacagct
     4981 aacaccacgt cgtccctatc tgctgcccta ggtctatgag tggttgctgg ataactttac
     5041 gggcatgcat aaggctcgta taatatattc agggagacca caacggtttc cctctacaaa
     5101 taattttgtt taacttttac tagagttcct tcctcgaaag gaaaaaaaaa atggctcgtg
     5161 tacagtttaa acaacgtgaa tctactgacg caatctttgt tcactgctcg gctaccaagc
     5221 caagtcagaa tgttggtgtc cgtgagattc gccagtggca caaagagcag ggttggctcg
     5281 atgtgggata ccactttatc atcaagcgag acggtactgt ggaggcagga cgagatgaga
     5341 tggctgtagg ctctcacgct aagggttaca accacaactc tatcggcgtc tgccttgttg
     5401 gtggtatcga cgataaaggt aagttcgacg ctaactttac gccagcccaa atgcaatccc
     5461 ttcgctcact gcttgtcaca ctgctggcta agtacgaagg cgctgtgctt cgcgcccatc
     5521 atgaggtggc gccgaaggct tccccttcgt tcgaccttaa gcgttggtgg gagaagaacg
     5581 aactggtcac ttctgaccgt ggtagcggcg gtggtgcgag tggtggtgcg gacatgggta
     5641 acgaaatcga cacccagaac cgtcagatcg accgtatcat ggaaaaagct gacggcggta
     5701 gcgcaggcag tggagcgggc ggtaacacga ttaacatcgc taagaacgac ttctctgaca
     5761 tcgaactggc tgctatcccg ttcaacactc tggctgacca ttacggtgag cgtttagctc
     5821 gcgaacagtt ggcccttgag catgagtctt acgagatggg tgaagcacgc ttccgcaaga
     5881 tgtttgagcg tcaacttaaa gctggtgagg ttgcggataa cgctgccgct aagcctctca
     5941 tcactaccct actccctaag atgattgcac gcatcaacga ctggtttgag gaagtgaaag
     6001 ctaagcgcgg caagcgcccg acagccttcc agttcctgca agaaatcaag ccggaagccg
     6061 tagcgtacat caccattaag accactctgg cttgcctaac cagtgctgac aatacaaccg
     6121 ttcaggctgt agcaagcgca atcggtcggg ccattgagga cgaggctcgc ttcggtcgta
     6181 tccgtgacct tgaagctaag cacttcaaga aaaacgttga ggaacaactc aacaagcgcg
     6241 tagggcacgt ctacaagaaa gcatttatgc aagttgtcga ggctgacatg ctctctaagg
     6301 gtctactcgg tggcgaggcg tggtcttcgt ggcataagga agactctatt catgtaggag
     6361 tacgctgcat cgagatgctc attgagtcaa ccggaatggt tagcttacac cgccaaaatg
     6421 ctggcgtagt aggtcaagac tctgagacta tcgaactcgc acctgaatac gctgaggcta
     6481 tcgcaacccg tgcaggtgcg ctggctggca tctctccgat gttccaacct tgcgtagttc
     6541 ctcctaagcc gtggactggc attactggtg gtggctattg ggctaacggt cgtcgtcctc
     6601 tggcgctggt gcgtactcac agtaagaaag cactgatgcg ctacgaagac gtttacatgc
     6661 ctgaggtgta caaagcgatt aacattgcgc aaaacaccgc atggaaaatc aacaagaaag
     6721 tcctagcggt cgccaacgta atcaccaagt ggaagcattg tccggtcgag gacatccctg
     6781 cgattgagcg tgaagaactc ccgatgaaac cggaagacat cgacatgaat cctgaggctc
     6841 tcaccgcgtg gaaacgtgct gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc
     6901 gccgtatcag ccttgagttc atgcttgagc aagccaataa gtttgctaac cataaggcca
     6961 tctggttccc ttacaacatg gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc
     7021 cgcaaggtaa cgatatgacc aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta
     7081 aggaaggtta ctactggctg aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc
     7141 cgttccctga gcgcatcaag ttcattgagg aaaaccacga gaacatcatg gcttgcgcta
     7201 agtctccact ggagaacact tggtgggctg agcaagattc tccgttctgc ttccttgcgt
     7261 tctgctttga gtacgctggg gtacagcacc acggcctgag ctataactgc tcccttccgc
     7321 tggcgtttga cgggtcttgc tctggcatcc agcacttctc cgcgatgctc cgagatgagg
     7381 taggtggtcg cgcggttaac ttgcttccta gtgaaaccgt tcaggacatc tacgggattg
     7441 ttgctaagaa agtcaacgag attctacaag cagacgcaat caatgggacc gataacgaag
     7501 tagttaccgt gaccgatgag aacactggtg aaatctctga gaaagtcaag ctgggcacta
     7561 aggcactggc tggtcaatgg ctggcttacg gtgttactcg cagtgtgact aagcgttcag
     7621 tcatgacgct ggcttacggg tccaaagagt tcggcttccg tcaacaagtg ctggaagata
     7681 ccattcagcc agctattgat tccggcaagg gtctgatgtt cactcagccg aatcaggctg
     7741 ctggatacat ggctaagctg atttgggaat ctgtgagcgt gacggtggta gctgcggttg
     7801 aagcaatgaa ctggcttaag tctgctgcta agctgctggc tgctgaggtc aaagataaga
     7861 agactggaga gattcttcgc aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc
     7921 ctgtgtggca ggaatacaag aagcctattc agacgcgctt gaacctgatg ttcctcggtc
     7981 agttccgctt acagcctacc attaacacca acaaagatag cgagattgat gcacacaaac
     8041 aggagtctgg tatcgctcct aactttgtac acagccaaga cggtagccac cttcgtaaga
     8101 ctgtagtgtg ggcacacgag aagtacggaa tcgaatcttt tgcactgatt cacgactcct
     8161 tcggtaccat tccggctgac gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg
     8221 acacatatga gtcttgtgat gtactggctg atttctacga ccagttcgct gaccagttgc
     8281 acgagtctca attggacaaa atgccagcac ttccggctaa aggtaacttg aacctccgtg
     8341 acatcttaga gtcggacttc gcgttcgcgt aatggagatt ttcaacatgc tccctcaatc
     8401 ggttgaatgt cgcccttttg tctttggcgc tggtaaacca tatgaatttt ctattgattg
     8461 tgacaaaata aacttattcc gtggtgtctt tgcgtttctt ttatatgttg ccacctttat
     8521 gtatgtattt tctacgtttg ctaacatact gcgtaataag gagtcttaat catgccagtt
     8581 cttttgggta ttccgtagaa aaaggaagag tatgagggaa gcggtgatcg ccgaagtatc
     8641 gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc
     8701 cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg atattgattt
     8761 gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct
     8821 tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat
     8881 tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg
     8941 agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga
     9001 tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc
     9061 ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt
     9121 aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt
     9181 gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga
     9241 ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctagacaggc
     9301 ttatcttgga caagaagaag atcgcttggc ctcg
//
