LOCUS       sequence-331972-        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-SNAP25d
FEATURES             Location/Qualifiers
     source          1..9335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(151..168)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(285..830)
                     /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(365..384)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1454..1473
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1454..1472
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1721..1738
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2749..3828
                     /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             3892..4872
                     /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             5155..5607
                     /codon_start=1
                     /product="lysozyme from bacteriophage T7"
                     /label=T7 lysozyme
                     /note="inhibitor of T7 RNA polymerase"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAVLRAHHEVAPKASPSFDLKRWWEKNELVTSDRG"
     CDS             join(8616..9335,1..69)
                     /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 tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta
       61 gtcggcaaaa cggatcgctt catgtggcag tagaaaaaag actgcaccgg tgcgtcagca
      121 gaatatgtga tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt
      181 tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag
      241 gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc
      301 ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga
      361 ctataaagat accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc
      421 tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac
      481 actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca
      541 gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc
      601 aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat
      661 gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt
      721 tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg
      781 ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc
      841 ttattaaggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
      901 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
      961 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     1021 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     1081 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     1141 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccgattcg agctcgcccg
     1201 gggatcgacc agttggtgat tttgaacttt tgctttgcca cggaacggtc tgcgttgtcg
     1261 ggaagatgcg tgatctgatc cttcaactca gcaaaagttc gatttattca acaaagccgc
     1321 cgtcccgtca agtcagcgta atgctctgcc agtgttacaa ccaattaaca agaggacatc
     1381 cggtcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgttttg ctgaggagac
     1441 ttagggaccc tactaatacg actcactata gggagaaaga aggagcgaca ttgctccgtg
     1501 tattcactcg ttggaatgaa tacacagtgc agtgtttatt ctgttattta tgccaaaaat
     1561 aaaggccact atcaggcagc tttgttgttc tgtttaccaa gttcaggagg taactcataa
     1621 gaaagacctg caggtgcagt aaaaaaagga aaaattgaaa aaattattat tcgcaattcc
     1681 tttagttgtt cctttctatt ctcactccgc tgaaactgtt catcaccatc accatcacgc
     1741 tgaaactgtt gaaagttgtt tagcaaaacc ccatacagaa aattcattta ctaacgtctg
     1801 gaaagacgac aaaactttag atcgttacgc taactatgag ggctgtctgt ggaatgctac
     1861 aggcgttgta gtttgtactg gtgacgaaac tcagtgttac ggtacatggg ttcctattgg
     1921 gcttgctatc cctgaaaatg agggtggtgg ctctgagggt ggcggttctg agggtggcgg
     1981 ttctgagggt ggcggtacta aacctcctga gtacggtgat acacctattc cgggctatac
     2041 ttatatcaac cctctcgacg gcacttatcc gcctggtact gagcaaaacc ccgctaatcc
     2101 taatccttct cttgaggagt ctcagcctct taatactttc atgtttcaga ataataggtt
     2161 ccgaaatagg cagggggcat taactgttta tacgggcact gttactcaag gcactgaccc
     2221 cgttaaaact tattaccagt acactcctgt atcatcaaaa gccatgtatg acgcttactg
     2281 gaacggtaaa ttcagagact gcgctttcca ttctggcttt aatgaggatc cattcgtttg
     2341 tgaatatcaa ggccaatcgt ctgacctgcc tcaacctcct gtcaatgctg gcggcggctc
     2401 tggtggtggt tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg
     2461 tggcggctct gagggaggcg gttccggtgg tggctcttcc caaatggctc aagtcggtga
     2521 cggtgataat tcacctttaa tgaataattt ccgtcaatat ttaccttccc tccctcaatc
     2581 ggttgaatgt cgcccttttg tctttggcgc tggtaaacct tacgagttca gtatcgactg
     2641 cgataagatc aacctgttcc gcggtgtctt tgcgtttctt ttatatgttg ccacctttat
     2701 gtatgtattt tctacgtttg ctaacatact gcgtaataag gagtcttaat gaaatttgga
     2761 aactttttgc ttacatacca acctccccaa ttttcccaaa cagaggtaat gaaacgtttg
     2821 gttaaattag gtcgcatctc tgaggagtgt ggttttgata ccgtatggtt actggagcat
     2881 catttcacgg agtttggttt gcttggtaac ccttatgtcg ctgctgcata tttacttggc
     2941 gcgactaaaa aattgaatgt aggaactgcc gctattgttc ttcccacagc ccatccagta
     3001 cgccaacttg aagatgtgaa tttattggat caaatgtcaa aaggacgatt tcggtttggt
     3061 atttgccgag ggctttacaa caaggacttt cgcgtattcg gcacagatat gaataacagt
     3121 cgcgccttag cggaatgctg gtacgggctg ataaagaatg gcatgacaga gggatatatg
     3181 gaagctgata atgaacatat caagttccat aaggtaaaag taaaccccgc ggcgtatagc
     3241 agaggtggcg caccggttta tgtggtggct gaatcagctt cgacgactga gtgggctgct
     3301 caatttggcc taccgatgat attaagttgg attataaata ctaacgaaaa gaaagcacaa
     3361 cttgagcttt ataatgaagt ggctcaagaa tatgggcacg atattcataa tatcgaccat
     3421 tgcttatcat atataacatc tgtagatcat gactcaatta aagcgaaaga gatttgccgg
     3481 aaatttctgg ggcattggta tgattcttat gtgaatgcta cgactatttt tgatgattca
     3541 gaccaaacaa gaggttatga tttcaataaa gggcagtggc gtgactttgt attaaaagga
     3601 cataaagata ctaatcgccg tattgattac agttacgaaa tcaatcccgt gggaacgccg
     3661 caggaatgta ttgacataat tcaaaaagac attgatgcta caggaatatc aaatatttgt
     3721 tgtggatttg aagctaatgg aacagtagac gaaattattg cttccatgaa gctcttccag
     3781 tctgatgtca tgccatttct taaagaaaaa caacgttcgc tattatatta tggcggtggc
     3841 ggtagcggcg gtggcggtag cggcggtggc ggtagcggcg gtggcggtag caaatttgga
     3901 ttgttcttcc ttaacttcat caattcaaca actgttcaag aacagagtat agttcgcatg
     3961 caggaaataa cggagtatgt tgataagttg aattttgaac agattttagt gtatgaaaat
     4021 catttttcag ataatggtgt tgtcggcgct cctctgactg tttctggttt tctgctcggt
     4081 ttaacagaga aaattaaaat tggttcatta aatcacatca ttacaactca tcatcctgtc
     4141 cgcatagcgg aggaagcttg cttattggat cagttaagtg aagggagatt tattttaggg
     4201 tttagtgatt gcgaaaaaaa agatgaaatg cattttttta atcgcccggt tgaatatcaa
     4261 cagcaactat ttgaagagtg ttatgaaatc attaacgatg ctttaacaac aggctattgt
     4321 aatccagata acgattttta tagcttccct aaaatatctg taaatcccca tgcttatacg
     4381 ccaggcggac ctcggaaata tgtaacagca accagtcatc atattgttga gtgggcggcc
     4441 aaaaaaggta ttcctctcat ctttaagtgg gatgattcta atgatgttag atatgaatat
     4501 gctgaaagat ataaagccgt tgcggataaa tatgacgttg acctatcaga gatagaccat
     4561 cagttaatga tattagttaa ctataacgaa gatagtaata aagctaaaca agagacgcgt
     4621 gcatttatta gtgattatgt tcttgaaatg caccctaatg aaaatttcga aaataaactt
     4681 gaagaaataa ttgcagaaaa cgctgtcgga aattatacgg agtgtataac tgcggctaag
     4741 ttggcaattg aaaagtgtgg tgcgaaaagt gtattgctgt cctttgaacc aatgaatgat
     4801 ttgatgagcc aaaaaaatgt aatcaatatt gttgatgata atattaagaa gtaccacacg
     4861 gaatatacct aaacttaatt aacggcactc ctcagcaaat ataatgaccc tcttgataac
     4921 ccaagagggc attttttaat gcccatggcg tcaattccga tttccggtat tctagagcac
     4981 agctaacacc acgtcgtccc tatctgctgc cctaggtcta tgagtggttg ctggataact
     5041 ttacgggcat gcataaggct cgtataatat attcagggag accacaacgg tttccctcta
     5101 caaataattt tgtttaactt ttactagagt tccttcctcg aaaggaaaaa aaaaatggct
     5161 cgtgtacagt ttaaacaacg tgaatctact gacgcaatct ttgttcactg ctcggctacc
     5221 aagccaagtc agaatgttgg tgtccgtgag attcgccagt ggcacaaaga gcagggttgg
     5281 ctcgatgtgg gataccactt tatcatcaag cgagacggta ctgtggaggc aggacgagat
     5341 gagatggctg taggctctca cgctaagggt tacaaccaca actctatcgg cgtctgcctt
     5401 gttggtggta tcgacgataa aggtaagttc gacgctaact ttacgccagc ccaaatgcaa
     5461 tcccttcgct cactgcttgt cacactgctg gctaagtacg aaggcgctgt gcttcgcgcc
     5521 catcatgagg tggcgccgaa ggcttcccct tcgttcgacc ttaagcgttg gtgggagaag
     5581 aacgaactgg tcacttctga ccgtggtagc ggcggtggtg cgagtggtgg tgcggacatg
     5641 ggtaacgaaa tcgacaccca gaaccgtcag atcgaccgta tcatggaaaa agctgacggc
     5701 ggtagcgcag gcagtggagc gggcggtaac acgattaaca tcgctaagaa cgacttctct
     5761 gacatcgaac tggctgctat cccgttcaac actctggctg accattacgg tgagcgttta
     5821 gctcgcgaac agttggccct tgagcatgag tcttacgaga tgggtgaagc acgcttccgc
     5881 aagatgtttg agcgtcaact taaagctggt gaggttgcgg ataacgctgc cgctaagcct
     5941 ctcatcacta ccctactccc taagatgatt gcacgcatca acgactggtt tgaggaagtg
     6001 aaagctaagc gcggcaagcg cccgacagcc ttccagttcc tgcaagaaat caagccggaa
     6061 gccgtagcgt acatcaccat taagaccact ctggcttgcc taaccagtgc tgacaataca
     6121 accgttcagg ctgtagcaag cgcaatcggt cgggccattg aggacgaggc tcgcttcggt
     6181 cgtatccgtg accttgaagc taagcacttc aagaaaaacg ttgaggaaca actcaacaag
     6241 cgcgtagggc acgtctacaa gaaagcattt atgcaagttg tcgaggctga catgctctct
     6301 aagggtctac tcggtggcga ggcgtggtct tcgtggcata aggaagactc tattcatgta
     6361 ggagtacgct gcatcgagat gctcattgag tcaaccggaa tggttagctt acaccgccaa
     6421 aatgctggcg tagtaggtca agactctgag actatcgaac tcgcacctga atacgctgag
     6481 gctatcgcaa cccgtgcagg tgcgctggct ggcatctctc cgatgttcca accttgcgta
     6541 gttcctccta agccgtggac tggcattact ggtggtggct attgggctaa cggtcgtcgt
     6601 cctctggcgc tggtgcgtac tcacagtaag aaagcactga tgcgctacga agacgtttac
     6661 atgcctgagg tgtacaaagc gattaacatt gcgcaaaaca ccgcatggaa aatcaacaag
     6721 aaagtcctag cggtcgccaa cgtaatcacc aagtggaagc attgtccggt cgaggacatc
     6781 cctgcgattg agcgtgaaga actcccgatg aaaccggaag acatcgacat gaatcctgag
     6841 gctctcaccg cgtggaaacg tgctgccgct gctgtgtacc gcaaggacaa ggctcgcaag
     6901 tctcgccgta tcagccttga gttcatgctt gagcaagcca ataagtttgc taaccataag
     6961 gccatctggt tcccttacaa catggactgg cgcggtcgtg tttacgctgt gtcaatgttc
     7021 aacccgcaag gtaacgatat gaccaaagga ctgcttacgc tggcgaaagg taaaccaatc
     7081 ggtaaggaag gttactactg gctgaaaatc cacggtgcaa actgtgcggg tgtcgataag
     7141 gttccgttcc ctgagcgcat caagttcatt gaggaaaacc acgagaacat catggcttgc
     7201 gctaagtctc cactggagaa cacttggtgg gctgagcaag attctccgtt ctgcttcctt
     7261 gcgttctgct ttgagtacgc tggggtacag caccacggcc tgagctataa ctgctccctt
     7321 ccgctggcgt ttgacgggtc ttgctctggc atccagcact tctccgcgat gctccgagat
     7381 gaggtaggtg gtcgcgcggt taacttgctt cctagtgaaa ccgttcagga catctacggg
     7441 attgttgcta agaaagtcaa cgagattcta caagcagacg caatcaatgg gaccgataac
     7501 gaagtagtta ccgtgaccga tgagaacact ggtgaaatct ctgagaaagt caagctgggc
     7561 actaaggcac tggctggtca atggctggct tacggtgtta ctcgcagtgt gactaagcgt
     7621 tcagtcatga cgctggctta cgggtccaaa gagttcggct tccgtcaaca agtgctggaa
     7681 gataccattc agccagctat tgattccggc aagggtctga tgttcactca gccgaatcag
     7741 gctgctggat acatggctaa gctgatttgg gaatctgtga gcgtgacggt ggtagctgcg
     7801 gttgaagcaa tgaactggct taagtctgct gctaagctgc tggctgctga ggtcaaagat
     7861 aagaagactg gagagattct tcgcaagcgt tgcgctgttc attgggtaac tcctgatggt
     7921 ttccctgtgt ggcaggaata caagaagcct attcagacgc gcttgaacct gatgttcctc
     7981 ggtcagttcc gcttacagcc taccattaac accaacaaag atagcgagat tgatgcacac
     8041 aaacaggagt ctggtatcgc tcctaacttt gtacacagcc aagacggtag ccaccttcgt
     8101 aagactgtag tgtgggcaca cgagaagtac ggaatcgaat cttttgcact gattcacgac
     8161 tccttcggta ccattccggc tgacgctgcg aacctgttca aagcagtgcg cgaaactatg
     8221 gttgacacat atgagtcttg tgatgtactg gctgatttct acgaccagtt cgctgaccag
     8281 ttgcacgagt ctcaattgga caaaatgcca gcacttccgg ctaaaggtaa cttgaacctc
     8341 cgtgacatct tagagtcgga cttcgcgttc gcgtaatgga gattttcaac atgctccctc
     8401 aatcggttga atgtcgccct tttgtctttg gcgctggtaa accatatgaa ttttctattg
     8461 attgtgacaa aataaactta ttccgtggtg tctttgcgtt tcttttatat gttgccacct
     8521 ttatgtatgt attttctacg tttgctaaca tactgcgtaa taaggagtct taatcatgcc
     8581 agttcttttg ggtattccgt agaaaaagga agagtatgag ggaagcggtg atcgccgaag
     8641 tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc
     8701 tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg
     8761 atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg
     8821 accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca
     8881 ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat
     8941 ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca
     9001 ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag
     9061 cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa
     9121 ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta
     9181 cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg
     9241 ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac
     9301 aggcttatct tggacaagaa gaagatcgct tggcc
//
