LOCUS       sequence-331959-        9389 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 9389)
  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 9389)
  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-VAMP1b
FEATURES             Location/Qualifiers
     source          1..9389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(92..109)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(226..771)
                     /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(306..325)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1660..1677
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2688..3767
                     /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             3831..4811
                     /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             5093..5542
                     /codon_start=1
                     /product="lysozyme from bacteriophage T7"
                     /label=T7 lysozyme
                     /note="inhibitor of T7 RNA polymerase"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAVLRAHHEVAPKASPSFDLKRWWEKNELVTSDR"
     CDS             join(8611..9389,1..10)
                     /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 agtcggcaaa acggatcgct tcatgtggca gtagaaaaaa gactgcaccg gtgcgtcagc
       61 agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg
      121 ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca
      181 ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg
      241 cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg
      301 actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc
      361 ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga
      421 cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc
      481 agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg
      541 caaaagcacc actggcagca gccactggta attgatttag aggagttagt cttgaagtca
      601 tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag
      661 ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg
      721 gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat
      781 cttattaagg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      841 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      901 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
      961 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1021 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1081 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccgattc gagctcgccc
     1141 ggggatcgac cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc
     1201 gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagccg
     1261 ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac aagaggacat
     1321 ccggtcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt gctgaggaga
     1381 cttagggacc ctactaatac gactcactat agggagaaag aaggagcgac attgctccgt
     1441 gtattcactc gttggaatga atacacagtg cagtgtttat tctgttattt atgccaaaaa
     1501 taaaggccac tatcaggcag ctttgttgtt ctgtttacca agttcaggag gtaactcata
     1561 agaaagacct gcaggtgcag taaaaaggaa aaaatgaaaa aattattatt cgcaattcct
     1621 ttagttgttc ctttctattc tcactccgct gaaactgttc atcaccatca ccatcacgct
     1681 gaaactgttg aaagttgttt agcaaaaccc catacagaaa attcatttac taacgtctgg
     1741 aaagacgaca aaactttaga tcgttacgct aactatgagg gctgtctgtg gaatgctaca
     1801 ggcgttgtag tttgtactgg tgacgaaact cagtgttacg gtacatgggt tcctattggg
     1861 cttgctatcc ctgaaaatga gggtggtggc tctgagggtg gcggttctga gggtggcggt
     1921 tctgagggtg gcggtactaa acctcctgag tacggtgata cacctattcc gggctatact
     1981 tatatcaacc ctctcgacgg cacttatccg cctggtactg agcaaaaccc cgctaatcct
     2041 aatccttctc ttgaggagtc tcagcctctt aatactttca tgtttcagaa taataggttc
     2101 cgaaataggc agggggcatt aactgtttat acgggcactg ttactcaagg cactgacccc
     2161 gttaaaactt attaccagta cactcctgta tcatcaaaag ccatgtatga cgcttactgg
     2221 aacggtaaat tcagagactg cgctttccat tctggcttta atgaggatcc attcgtttgt
     2281 gaatatcaag gccaatcgtc tgacctgcct caacctcctg tcaatgctgg cggcggctct
     2341 ggtggtggtt ctggtggcgg ctctgagggt ggtggctctg agggtggcgg ttctgagggt
     2401 ggcggctctg agggaggcgg ttccggtggt ggctcttccc aaatggctca agtcggtgac
     2461 ggtgataatt cacctttaat gaataatttc cgtcaatatt taccttccct ccctcaatcg
     2521 gttgaatgtc gcccttttgt ctttggcgct ggtaaacctt acgagttcag tatcgactgc
     2581 gataagatca acctgttccg cggtgtcttt gcgtttcttt tatatgttgc cacctttatg
     2641 tatgtatttt ctacgtttgc taacatactg cgtaataagg agtcttaatg aaatttggaa
     2701 actttttgct tacataccaa cctccccaat tttcccaaac agaggtaatg aaacgtttgg
     2761 ttaaattagg tcgcatctct gaggagtgtg gttttgatac cgtatggtta ctggagcatc
     2821 atttcacgga gtttggtttg cttggtaacc cttatgtcgc tgctgcatat ttacttggcg
     2881 cgactaaaaa attgaatgta ggaactgccg ctattgttct tcccacagcc catccagtac
     2941 gccaacttga agatgtgaat ttattggatc aaatgtcaaa aggacgattt cggtttggta
     3001 tttgccgagg gctttacaac aaggactttc gcgtattcgg cacagatatg aataacagtc
     3061 gcgccttagc ggaatgctgg tacgggctga taaagaatgg catgacagag ggatatatgg
     3121 aagctgataa tgaacatatc aagttccata aggtaaaagt aaaccccgcg gcgtatagca
     3181 gaggtggcgc accggtttat gtggtggctg aatcagcttc gacgactgag tgggctgctc
     3241 aatttggcct accgatgata ttaagttgga ttataaatac taacgaaaag aaagcacaac
     3301 ttgagcttta taatgaagtg gctcaagaat atgggcacga tattcataat atcgaccatt
     3361 gcttatcata tataacatct gtagatcatg actcaattaa agcgaaagag atttgccgga
     3421 aatttctggg gcattggtat gattcttatg tgaatgctac gactattttt gatgattcag
     3481 accaaacaag aggttatgat ttcaataaag ggcagtggcg tgactttgta ttaaaaggac
     3541 ataaagatac taatcgccgt attgattaca gttacgaaat caatcccgtg ggaacgccgc
     3601 aggaatgtat tgacataatt caaaaagaca ttgatgctac aggaatatca aatatttgtt
     3661 gtggatttga agctaatgga acagtagacg aaattattgc ttccatgaag ctcttccagt
     3721 ctgatgtcat gccatttctt aaagaaaaac aacgttcgct attatattat ggcggtggcg
     3781 gtagcggcgg tggcggtagc ggcggtggcg gtagcggcgg tggcggtagc aaatttggat
     3841 tgttcttcct taacttcatc aattcaacaa ctgttcaaga acagagtata gttcgcatgc
     3901 aggaaataac ggagtatgtt gataagttga attttgaaca gattttagtg tatgaaaatc
     3961 atttttcaga taatggtgtt gtcggcgctc ctctgactgt ttctggtttt ctgctcggtt
     4021 taacagagaa aattaaaatt ggttcattaa atcacatcat tacaactcat catcctgtcc
     4081 gcatagcgga ggaagcttgc ttattggatc agttaagtga agggagattt attttagggt
     4141 ttagtgattg cgaaaaaaaa gatgaaatgc atttttttaa tcgcccggtt gaatatcaac
     4201 agcaactatt tgaagagtgt tatgaaatca ttaacgatgc tttaacaaca ggctattgta
     4261 atccagataa cgatttttat agcttcccta aaatatctgt aaatccccat gcttatacgc
     4321 caggcggacc tcggaaatat gtaacagcaa ccagtcatca tattgttgag tgggcggcca
     4381 aaaaaggtat tcctctcatc tttaagtggg atgattctaa tgatgttaga tatgaatatg
     4441 ctgaaagata taaagccgtt gcggataaat atgacgttga cctatcagag atagaccatc
     4501 agttaatgat attagttaac tataacgaag atagtaataa agctaaacaa gagacgcgtg
     4561 catttattag tgattatgtt cttgaaatgc accctaatga aaatttcgaa aataaacttg
     4621 aagaaataat tgcagaaaac gctgtcggaa attatacgga gtgtataact gcggctaagt
     4681 tggcaattga aaagtgtggt gcgaaaagtg tattgctgtc ctttgaacca atgaatgatt
     4741 tgatgagcca aaaaaatgta atcaatattg ttgatgataa tattaagaag taccacacgg
     4801 aatataccta aacttaatta acggcactcc tcagcaaata taatgaccct cttgataacc
     4861 caagagggca ttttttaatg cccatggcgt caattccgat ttccggtatt ctagagcaca
     4921 gctaacacca cgtcgtccct atctgctgcc ctaggtctat gagtggttgc tggataactt
     4981 tacgggcatg cataaggctc gtataatata ttcagggaga ccacaacggt ttccctctac
     5041 aaataatttt gtttaacttt tactagagtt ccttcctcga aaggaaaaaa aaatggctcg
     5101 tgtacagttt aaacaacgtg aatctactga cgcaatcttt gttcactgct cggctaccaa
     5161 gccaagtcag aatgttggtg tccgtgagat tcgccagtgg cacaaagagc agggttggct
     5221 cgatgtggga taccacttta tcatcaagcg agacggtact gtggaggcag gacgagatga
     5281 gatggctgta ggctctcacg ctaagggtta caaccacaac tctatcggcg tctgccttgt
     5341 tggtggtatc gacgataaag gtaagttcga cgctaacttt acgccagccc aaatgcaatc
     5401 ccttcgctca ctgcttgtca cactgctggc taagtacgaa ggcgctgtgc ttcgcgccca
     5461 tcatgaggtg gcgccgaagg cttccccttc gttcgacctt aagcgttggt gggagaagaa
     5521 cgaactggtc acatctgacc gtacctctaa ccgtcgtctg cagcagaccc aggctcaggt
     5581 tgaagaagtt gttgacatca tccgtgttaa cgttgacaaa gttctggaac gtgaccagaa
     5641 actgtctgaa ctggacgacc gtgctgacgc tctgcaggct ggtgcttctc agttcgaatc
     5701 ttctgctgct aaactgaaac 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 tgtgcattgg 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 tcgcgcgcag atcagttgga agaatttgtc
     9361 cactacgtga aaggcgagat caccaaggt
//
