LOCUS       sequence-244412-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AO"" to be used 
            for allelic exchange on bacterial chromosomes and subsequent 
            molecular identification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4728)
  AUTHORS   Shaw JA, Bourret TJ
  TITLE     Digital PCR-based Competitive Index for High-throughput Analysis of 
            Fitness in Salmonella.
  JOURNAL   J Vis Exp. 2019 May 13;(147). doi: 10.3791/59630.
  PUBMED    31132072
REFERENCE   2  (bases 1 to 4728)
  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"; journalName: "J Vis 
            Exp."; date: "2019-05-13"; volume: "(147). doi"; pages: " 
            10.3791/59630"
COMMENT     Sequence Label: pSKAP_AO
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    93..114
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(129..134,135..152,153..159)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    167..183
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     172..194
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     191..207
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     191..207
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     226..246
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        228..246
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    316..349
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             717..1511
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(771..790)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1381..1400
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1524..1571
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     complement(1661..1677)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1662..1678)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1702..1721)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1703..1721)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1731..1748)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1731..1747)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1740..1762)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1889..2344
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1976..1995)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2186..2207
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2370..2474
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2963..3622)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3537..3556
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3623..3725)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3945..4533
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4434..4453
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4687..4704
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
       61 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
      121 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      181 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
      241 taaagggaac aaaagctgga gctccaccgc ggtggcggcc gctctagccc gggcggatcc
      301 gtcgacctgc agttcgaagt tcctattctc tagaaagtat aggaacttca gagcgctttt
      361 gaagctcacg ctgccgcaag cactcagggc gcaagggctg ctaaaggaag cggaacacgt
      421 agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct
      481 ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc
      541 gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc
      601 cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga
      661 tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga
      721 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
      781 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
      841 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
      901 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
      961 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     1021 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     1081 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     1141 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     1201 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     1261 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     1321 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     1381 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     1441 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     1501 agttcttcta ataaggggat cttgaagttc ctattccgaa gttcctattc tctagaaagt
     1561 ataggaactt cgaagcagct ccagcctaca caatcggtcc acaaccacac tcagtgatac
     1621 tctcaagacg tgtaatgctg caatctgcat gcaagcttat cgataccgtc gacctcgagg
     1681 gggggcccgg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     1741 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     1801 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     1861 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1921 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1981 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2041 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2101 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2161 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2221 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2281 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2341 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2401 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2461 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2521 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2581 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2641 agagttttcg ccccgaagaa cgttttccaa tgatgagcac tttttgatca gaaaaaaagg
     2701 atcatatcgt caattattac ctccacgggg agagcctgag caaactggcc tcaggcattt
     2761 gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca
     2821 taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat
     2881 ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc gtttaagggc
     2941 accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta
     3001 attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg
     3061 ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg
     3121 cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat
     3181 tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac
     3241 cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt
     3301 cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa
     3361 cactatccca tatcaccagc tcaccgtctt tcattgccat acgtaattcc ggatgagcat
     3421 tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta
     3481 cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa
     3541 ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat
     3601 atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc gacaactcaa
     3661 aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc
     3721 gatcaacgtc tcattttcgc caaaagttgg cccagggctt cccggtatca acagggacac
     3781 caggatttat ttattctgcg aagtgatctt ccgtcacagg tatttattcg gtcgaaaagg
     3841 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3901 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3961 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4021 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4081 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4141 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4201 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4261 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4321 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4381 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4441 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4501 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4561 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4621 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4681 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgc
//
