LOCUS       sequence-239245-        4727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AM"" 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 4727)
  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 4727)
  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_AM
FEATURES             Location/Qualifiers
     source          1..4727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(775..822)
                     /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)."
     CDS             complement(835..1629)
                     /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(946..965)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1556..1575
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     protein_bind    complement(1997..2030)
                     /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)"
     primer_bind     complement(2100..2120)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2100..2118)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2139..2155)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2139..2155)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2152..2174)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2163..2179
                     /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)."
     promoter        complement(join(2187..2193,2194..2211,2212..2217))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2232..2253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2370..2387)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2541..3129)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2621..2640)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3349..3451
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3452..4111
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3518..3537)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4599..4704)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgcatgc agattgcagc attacacgtc
      721 ttgagaacac tgtgtggtag agctagtgcc gattgtgtag gctggagctg cttcgaagtt
      781 cctatacttt ctagagaata ggaacttcgg aataggaact tcaagatccc cttattagaa
      841 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
      901 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
      961 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     1021 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     1081 atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc
     1141 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     1201 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
     1261 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
     1321 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
     1381 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
     1441 ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
     1501 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
     1561 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
     1621 ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca
     1681 tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc
     1741 agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg cccagtctag
     1801 ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg cgttttccct
     1861 tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct gcggactggc
     1921 tttctacgtg ttccgcttcc tttagcagcc cttgcgccct gagtgcttgc ggcagcgtga
     1981 gcttcaaaag cgctctgaag ttcctatact ttctagagaa taggaacttc gaactgcagg
     2041 tcgacggatc cgcccgggct agagcggccg ccaccgcggt ggagctccag cttttgttcc
     2101 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     2161 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2221 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2281 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2341 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2401 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2461 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2521 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2581 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2641 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2701 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2761 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2821 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2881 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2941 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     3001 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3061 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3121 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3181 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatccttttc
     3241 gaccgaataa atacctgtga cggaagatca cttcgcagaa taaataaatc ctggtgtccc
     3301 tgttgatacc gggaagccct gggccaactt ttggcgaaaa tgagacgttg atcggcacgt
     3361 aagaggttcc aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt
     3421 gtcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac
     3481 cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca
     3541 atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa
     3601 aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca
     3661 tccggaatta cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc
     3721 ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca
     3781 cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa
     3841 cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg
     3901 ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt
     3961 tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca
     4021 ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca
     4081 gtactgcgat gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta
     4141 aacgcctggt tgctacgcct gaataagtga taataagcgg atgaatggca gaaattcgaa
     4201 agcaaattcg acccggtcgt cggttcaggg cagggtcgtt aaatagccgc ttatgtctat
     4261 tgctggttta ccggtttatt gactaccgga agcagtgtga ccgtgtgctt ctcaaatgcc
     4321 tgaggccagt ttgctcaggc tctccccgtg gaggtaataa ttgacgatat gatccttttt
     4381 ttctgatcaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4441 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4501 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4561 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4621 tgaagcattt atcaagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4681 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
