LOCUS       sequence-245013-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AN"" 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_AN
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    109..130
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(145..150,151..168,169..175)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    183..199
                     /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     188..210
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     207..223
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     207..223
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     242..262
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        244..262
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    332..365
                     /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             733..1527
                     /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(787..806)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1397..1416
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1540..1587
                     /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(1677..1693)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1678..1694)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1718..1737)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1719..1737)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1747..1764)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1747..1763)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1756..1778)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1905..2360
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1992..2011)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2202..2223
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2386..2490
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2979..3638)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3553..3572
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3639..3741)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3961..4549
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4450..4469
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4703..4720
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
       61 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      121 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      181 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      241 cgcaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc
      301 tagcccgggc ggatccgtcg acctgcagtt cgaagttcct attctctaga aagtatagga
      361 acttcagagc gcttttgaag ctcacgctgc cgcaagcact cagggcgcaa gggctgctaa
      421 aggaagcgga acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
      481 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
      541 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
      601 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
      661 ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga
      721 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
      781 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
      841 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
      901 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
      961 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     1021 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     1081 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     1141 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     1201 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     1261 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     1321 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     1381 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     1441 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     1501 tatcgccttc ttgacgagtt cttctaataa ggggatcttg aagttcctat tccgaagttc
     1561 ctattctcta gaaagtatag gaacttcgaa gcagctccag cctacacaat cgcgcacatg
     1621 ctctacgaga cagtcttctc aagacgtgta atgctgcaat ctgcatgcaa gcttatcgat
     1681 accgtcgacc tcgagggggg gcccggtacc caattcgccc tatagtgagt cgtattacgc
     1741 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     1801 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     1861 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     1921 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1981 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2041 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2101 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2161 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2221 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2281 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2341 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2401 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2461 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     2521 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     2581 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     2641 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     2701 tgatcagaaa aaaaggatca tatcgtcaat tattacctcc acggggagag cctgagcaaa
     2761 ctggcctcag gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt
     2821 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc
     2881 gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa
     2941 ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca
     3001 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca
     3061 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc
     3121 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg
     3181 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
     3241 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg
     3301 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg
     3361 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgt
     3421 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg
     3481 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
     3541 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat
     3601 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa
     3661 aatctcgaca actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg
     3721 gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg
     3781 gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt
     3841 tattcggtcg aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3901 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3961 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4021 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4081 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4141 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4201 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4261 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4321 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4381 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4441 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4501 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4561 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4621 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4681 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcgga
//
