LOCUS       sequence-331952-        9390 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 9390)
  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 9390)
  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-VAMP1c
FEATURES             Location/Qualifiers
     source          1..9390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(110..127)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(244..789)
                     /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(324..343)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1413..1432
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1413..1431
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1679..1696
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2707..3786
                     /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             3850..4830
                     /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             5112..5561
                     /codon_start=1
                     /product="lysozyme from bacteriophage T7"
                     /label=T7 lysozyme
                     /note="inhibitor of T7 RNA polymerase"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAVLRAHHEVAPKASPSFDLKRWWEKNELVTSDR"
     CDS             join(8630..9390,1..28)
                     /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 aggcgagatc accaaggtag tcggcaaaac ggatcgcttc atgtggcagt agaaaaaaga
       61 ctgcaccggt gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg
      121 actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag
      181 atttcctgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc
      241 gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg
      301 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt
      361 gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt
      421 gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta
      481 tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt
      541 ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag
      601 gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac
      661 tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa
      721 aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa
      781 cgatctcaag aagatcatct tattaagggg tctgacgctc agtggaacga aaactcacgt
      841 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      901 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      961 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1021 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1081 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1141 gccgattcga gctcgcccgg ggatcgacca gttggtgatt ttgaactttt gctttgccac
     1201 ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg
     1261 atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac
     1321 caattaacaa gaggacatcc ggtcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     1381 ttcgttttgc tgaggagact tagggaccct actaatacga ctcactatag ggagaaagaa
     1441 ggagcgacat tgctccgtgt attcactcgt tggaatgaat acacagtgca gtgtttattc
     1501 tgttatttat gccaaaaata aaggccacta tcaggcagct ttgttgttct gtttaccaag
     1561 ttcaggaggt aactcataag aaagacctgc agggcagtaa aaaaaaagga aaatgaaaaa
     1621 attattattc gcaattcctt tagttgttcc tttctattct cactccgctg aaactgttca
     1681 tcaccatcac catcacgctg aaactgttga aagttgttta gcaaaacccc atacagaaaa
     1741 ttcatttact aacgtctgga aagacgacaa aactttagat cgttacgcta actatgaggg
     1801 ctgtctgtgg aatgctacag gcgttgtagt ttgtactggt gacgaaactc agtgttacgg
     1861 tacatgggtt cctattgggc ttgctatccc tgaaaatgag ggtggtggct ctgagggtgg
     1921 cggttctgag ggtggcggtt ctgagggtgg cggtactaaa cctcctgagt acggtgatac
     1981 acctattccg ggctatactt atatcaaccc tctcgacggc acttatccgc ctggtactga
     2041 gcaaaacccc gctaatccta atccttctct tgaggagtct cagcctctta atactttcat
     2101 gtttcagaat aataggttcc gaaataggca gggggcatta actgtttata cgggcactgt
     2161 tactcaaggc actgaccccg ttaaaactta ttaccagtac actcctgtat catcaaaagc
     2221 catgtatgac gcttactgga acggtaaatt cagagactgc gctttccatt ctggctttaa
     2281 tgaggatcca ttcgtttgtg aatatcaagg ccaatcgtct gacctgcctc aacctcctgt
     2341 caatgctggc ggcggctctg gtggtggttc tggtggcggc tctgagggtg gtggctctga
     2401 gggtggcggt tctgagggtg gcggctctga gggaggcggt tccggtggtg gctcttccca
     2461 aatggctcaa gtcggtgacg gtgataattc acctttaatg aataatttcc gtcaatattt
     2521 accttccctc cctcaatcgg ttgaatgtcg cccttttgtc tttggcgctg gtaaacctta
     2581 cgagttcagt atcgactgcg ataagatcaa cctgttccgc ggtgtctttg cgtttctttt
     2641 atatgttgcc acctttatgt atgtattttc tacgtttgct aacatactgc gtaataagga
     2701 gtcttaatga aatttggaaa ctttttgctt acataccaac ctccccaatt ttcccaaaca
     2761 gaggtaatga aacgtttggt taaattaggt cgcatctctg aggagtgtgg ttttgatacc
     2821 gtatggttac tggagcatca tttcacggag tttggtttgc ttggtaaccc ttatgtcgct
     2881 gctgcatatt tacttggcgc gactaaaaaa ttgaatgtag gaactgccgc tattgttctt
     2941 cccacagccc atccagtacg ccaacttgaa gatgtgaatt tattggatca aatgtcaaaa
     3001 ggacgatttc ggtttggtat ttgccgaggg ctttacaaca aggactttcg cgtattcggc
     3061 acagatatga ataacagtcg cgccttagcg gaatgctggt acgggctgat aaagaatggc
     3121 atgacagagg gatatatgga agctgataat gaacatatca agttccataa ggtaaaagta
     3181 aaccccgcgg cgtatagcag aggtggcgca ccggtttatg tggtggctga atcagcttcg
     3241 acgactgagt gggctgctca atttggccta ccgatgatat taagttggat tataaatact
     3301 aacgaaaaga aagcacaact tgagctttat aatgaagtgg ctcaagaata tgggcacgat
     3361 attcataata tcgaccattg cttatcatat ataacatctg tagatcatga ctcaattaaa
     3421 gcgaaagaga tttgccggaa atttctgggg cattggtatg attcttatgt gaatgctacg
     3481 actatttttg atgattcaga ccaaacaaga ggttatgatt tcaataaagg gcagtggcgt
     3541 gactttgtat taaaaggaca taaagatact aatcgccgta ttgattacag ttacgaaatc
     3601 aatcccgtgg gaacgccgca ggaatgtatt gacataattc aaaaagacat tgatgctaca
     3661 ggaatatcaa atatttgttg tggatttgaa gctaatggaa cagtagacga aattattgct
     3721 tccatgaagc tcttccagtc tgatgtcatg ccatttctta aagaaaaaca acgttcgcta
     3781 ttatattatg gcggtggcgg tagcggcggt ggcggtagcg gcggtggcgg tagcggcggt
     3841 ggcggtagca aatttggatt gttcttcctt aacttcatca attcaacaac tgttcaagaa
     3901 cagagtatag ttcgcatgca ggaaataacg gagtatgttg ataagttgaa ttttgaacag
     3961 attttagtgt atgaaaatca tttttcagat aatggtgttg tcggcgctcc tctgactgtt
     4021 tctggttttc tgctcggttt aacagagaaa attaaaattg gttcattaaa tcacatcatt
     4081 acaactcatc atcctgtccg catagcggag gaagcttgct tattggatca gttaagtgaa
     4141 gggagattta ttttagggtt tagtgattgc gaaaaaaaag atgaaatgca tttttttaat
     4201 cgcccggttg aatatcaaca gcaactattt gaagagtgtt atgaaatcat taacgatgct
     4261 ttaacaacag gctattgtaa tccagataac gatttttata gcttccctaa aatatctgta
     4321 aatccccatg cttatacgcc aggcggacct cggaaatatg taacagcaac cagtcatcat
     4381 attgttgagt gggcggccaa aaaaggtatt cctctcatct ttaagtggga tgattctaat
     4441 gatgttagat atgaatatgc tgaaagatat aaagccgttg cggataaata tgacgttgac
     4501 ctatcagaga tagaccatca gttaatgata ttagttaact ataacgaaga tagtaataaa
     4561 gctaaacaag agacgcgtgc atttattagt gattatgttc ttgaaatgca ccctaatgaa
     4621 aatttcgaaa ataaacttga agaaataatt gcagaaaacg ctgtcggaaa ttatacggag
     4681 tgtataactg cggctaagtt ggcaattgaa aagtgtggtg cgaaaagtgt attgctgtcc
     4741 tttgaaccaa tgaatgattt gatgagccaa aaaaatgtaa tcaatattgt tgatgataat
     4801 attaagaagt accacacgga atatacctaa acttaattaa cggcactcct cagcaaatat
     4861 aatgaccctc ttgataaccc aagagggcat tttttaatgc ccatggcgtc aattccgatt
     4921 tccggtattc tagagcacag ctaacaccac gtcgtcccta tctgctgccc taggtctatg
     4981 agtggttgct ggataacttt acgggcatgc ataaggctcg tataatatat tcagggagac
     5041 cacaacggtt tccctctaca aataattttg tttaactttt actagagttc cttcctcgaa
     5101 aggaaaaaaa aatggctcgt gtacagttta aacaacgtga atctactgac gcaatctttg
     5161 ttcactgctc ggctaccaag ccaagtcaga atgttggtgt ccgtgagatt cgccagtggc
     5221 acaaagagca gggttggctc gatgtgggat accactttat catcaagcga gacggtactg
     5281 tggaggcagg acgagatgag atggctgtag gctctcacgc taagggttac aaccacaact
     5341 ctatcggcgt ctgccttgtt ggtggtatcg acgataaagg taagttcgac gctaacttta
     5401 cgccagccca aatgcaatcc cttcgctcac tgcttgtcac actgctggct aagtacgaag
     5461 gcgctgtgct tcgcgcccat catgaggtgg cgccgaaggc ttccccttcg ttcgacctta
     5521 agcgttggtg ggagaagaac gaactggtca catctgaccg tacctctaac cgtcgtctgc
     5581 agcagaccca ggctcaggtt gaagaagttg ttgacatcat ccgtgttaac gttgacaaag
     5641 ttctggaacg tgaccagaaa ctgtctgaac tggacgaccg tgctgacgct ctgcaggctg
     5701 gtgcttctca gttcgaatct tctgctgcta aactgaaacg taacacgatt aacatcgcta
     5761 agaacgactt ctctgacatc gaactggctg ctatcccgtt caacactctg gctgaccatt
     5821 acggtgagcg tttagctcgc gaacagttgg cccttgagca tgagtcttac gagatgggtg
     5881 aagcacgctt ccgcaagatg tttgagcgtc aacttaaagc tggtgaggtt gcggataacg
     5941 ctgccgctaa gcctctcatc actaccctac tccctaagat gattgcacgc atcaacgact
     6001 ggtttgagga agtgaaagct aagcgcggca agcgcccgac agccttccag ttcctgcaag
     6061 aaatcaagcc ggaagccgta gcgtacatca ccattaagac cactctggct tgcctaacca
     6121 gtgctgacaa tacaaccgtt caggctgtag caagcgcaat cggtcgggcc attgaggacg
     6181 aggctcgctt cggtcgtatc cgtgaccttg aagctaagca cttcaagaaa aacgttgagg
     6241 aacaactcaa caagcgcgta gggcacgtct acaagaaagc atttatgcaa gttgtcgagg
     6301 ctgacatgct ctctaagggt ctactcggtg gcgaggcgtg gtcttcgtgg cataaggaag
     6361 actctattca tgtaggagta cgctgcatcg agatgctcat tgagtcaacc ggaatggtta
     6421 gcttacaccg ccaaaatgct ggcgtagtag gtcaagactc tgagactatc gaactcgcac
     6481 ctgaatacgc tgaggctatc gcaacccgtg caggtgcgct ggctggcatc tctccgatgt
     6541 tccaaccttg cgtagttcct cctaagccgt ggactggcat tactggtggt ggctattggg
     6601 ctaacggtcg tcgtcctctg gcgctggtgc gtactcacag taagaaagca ctgatgcgct
     6661 acgaagacgt ttacatgcct gaggtgtaca aagcgattaa cattgcgcaa aacaccgcat
     6721 ggaaaatcaa caagaaagtc ctagcggtcg ccaacgtaat caccaagtgg aagcattgtc
     6781 cggtcgagga catccctgcg attgagcgtg aagaactccc gatgaaaccg gaagacatcg
     6841 acatgaatcc tgaggctctc accgcgtgga aacgtgctgc cgctgctgtg taccgcaagg
     6901 acaaggctcg caagtctcgc cgtatcagcc ttgagttcat gcttgagcaa gccaataagt
     6961 ttgctaacca taaggccatc tggttccctt acaacatgga ctggcgcggt cgtgtttacg
     7021 ctgtgtcaat gttcaacccg caaggtaacg atatgaccaa aggactgctt acgctggcga
     7081 aaggtaaacc aatcggtaag gaaggttact actggctgaa aatccacggt gcaaactgtg
     7141 cgggtgtcga taaggttccg ttccctgagc gcatcaagtt cattgaggaa aaccacgaga
     7201 acatcatggc ttgcgctaag tctccactgg agaacacttg gtgggctgag caagattctc
     7261 cgttctgctt ccttgcgttc tgctttgagt acgctggggt acagcaccac ggcctgagct
     7321 ataactgctc ccttccgctg gcgtttgacg ggtcttgctc tggcatccag cacttctccg
     7381 cgatgctccg agatgaggta ggtggtcgcg cggttaactt gcttcctagt gaaaccgttc
     7441 aggacatcta cgggattgtt gctaagaaag tcaacgagat tctacaagca gacgcaatca
     7501 atgggaccga taacgaagta gttaccgtga ccgatgagaa cactggtgaa atctctgaga
     7561 aagtcaagct gggcactaag gcactggctg gtcaatggct ggcttacggt gttactcgca
     7621 gtgtgactaa gcgttcagtc atgacgctgg cttacgggtc caaagagttc ggcttccgtc
     7681 aacaagtgct ggaagatacc attcagccag ctattgattc cggcaagggt ctgatgttca
     7741 ctcagccgaa tcaggctgct ggatacatgg ctaagctgat ttgggaatct gtgagcgtga
     7801 cggtggtagc tgcggttgaa gcaatgaact ggcttaagtc tgctgctaag ctgctggctg
     7861 ctgaggtcaa agataagaag actggagaga ttcttcgcaa gcgttgcgct gtgcattggg
     7921 taactcctga tggtttccct gtgtggcagg aatacaagaa gcctattcag acgcgcttga
     7981 acctgatgtt cctcggtcag ttccgcttac agcctaccat taacaccaac aaagatagcg
     8041 agattgatgc acacaaacag gagtctggta tcgctcctaa ctttgtacac agccaagacg
     8101 gtagccacct tcgtaagact gtagtgtggg cacacgagaa gtacggaatc gaatcttttg
     8161 cactgattca cgactccttc ggtaccattc cggctgacgc tgcgaacctg ttcaaagcag
     8221 tgcgcgaaac tatggttgac acatatgagt cttgtgatgt actggctgat ttctacgacc
     8281 agtttgctga ccagttgcac gagtctcaat tggacaaaat gccagcactt ccggctaaag
     8341 gtaacttgaa cctccgtgac atcttagagt cggacttcgc gttcgcgtaa tggagatttt
     8401 caacatgctc cctcaatcgg ttgaatgtcg cccttttgtc tttggcgctg gtaaaccata
     8461 tgaattttct attgattgtg acaaaataaa cttattccgt ggtgtctttg cgtttctttt
     8521 atatgttgcc acctttatgt atgtattttc tacgtttgct aacatactgc gtaataagga
     8581 gtcttaatca tgccagttct tttgggtatt ccgtagaaaa aggaagagta tgagggaagc
     8641 ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct
     8701 cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc
     8761 acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg
     8821 agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg
     8881 cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa
     8941 gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc
     9001 agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc
     9061 cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga
     9121 ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg
     9181 aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc
     9241 gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat
     9301 acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga
     9361 tcagttggaa gaatttgtcc actacgtgaa
//
