LOCUS       sequence-245021-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AT"" 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_AT
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    89..110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(125..130,131..148,149..155)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..179
                     /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     168..190
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     187..203
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     187..203
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     222..242
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        224..242
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    312..345
                     /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             713..1507
                     /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(767..786)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1377..1396
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1520..1567
                     /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(1657..1673)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1658..1674)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1698..1717)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1699..1717)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1727..1744)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1727..1743)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1736..1758)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1885..2340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1972..1991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2182..2203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2366..2470
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2959..3618)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3533..3552
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3619..3721)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3941..4529
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4430..4449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4683..4700
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
       61 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
      121 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      181 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
      241 gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagcccgggc ggatccgtcg
      301 acctgcagtt cgaagttcct attctctaga aagtatagga acttcagagc gcttttgaag
      361 ctcacgctgc cgcaagcact cagggcgcaa gggctgctaa aggaagcgga acacgtagaa
      421 agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg ctatctggac
      481 aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta catggcgata
      541 gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg gggcgccctc
      601 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
      661 atggcgcagg ggatcaagat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
      721 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
      781 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
      841 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
      901 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
      961 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     1021 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     1081 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     1141 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     1201 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     1261 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     1321 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     1381 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     1441 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     1501 cttctaataa ggggatcttg aagttcctat tccgaagttc ctattctcta gaaagtatag
     1561 gaacttcgaa gcagctccag cctacacaat cggtctagcc atgtcacgga cactggtctc
     1621 aagacgtgta atgctgcaat ctgcatgcaa gcttatcgat accgtcgacc tcgagggggg
     1681 gcccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     1741 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     1801 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     1861 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1921 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1981 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2041 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2101 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2161 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2221 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2281 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2341 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2401 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2461 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2521 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2581 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2641 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt tgatcagaaa aaaaggatca
     2701 tatcgtcaat tattacctcc acggggagag cctgagcaaa ctggcctcag gcatttgaga
     2761 agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag
     2821 cggctattta acgaccctgc cctgaaccga cgaccgggtc gaatttgctt tcgaatttct
     2881 gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt aagggcacca
     2941 ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc
     3001 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     3061 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     3121 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     3181 tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     3241 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     3301 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     3361 atcccatatc accagctcac cgtctttcat tgccatacgt aattccggat gagcattcat
     3421 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     3481 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     3541 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     3601 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgaca actcaaaaaa
     3661 tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc
     3721 aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg
     3781 atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggtcg aaaaggatct
     3841 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3901 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3961 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4021 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4081 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4141 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4201 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4261 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4321 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4381 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4441 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4501 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4561 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4621 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4681 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaac
//
