LOCUS       sequence-244411-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AI"" 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_AI
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..91
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     325..342
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    459..480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(495..500,501..518,519..525)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    533..549
                     /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     538..560
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     557..573
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     557..573
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     592..612
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        594..612
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    682..715
                     /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             1083..1877
                     /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(1137..1156)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1747..1766
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1890..1937
                     /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(2027..2043)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2028..2044)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2068..2087)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2069..2087)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2097..2114)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2097..2113)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2106..2128)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2255..2710
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2342..2361)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2552..2573
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2736..2840
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3329..3988)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3903..3922
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3989..4091)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      join(4311..4728,1..171)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
       61 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      121 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      181 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      241 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      301 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      361 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      421 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      481 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      541 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      601 cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagcccgggc
      661 ggatccgtcg acctgcagtt cgaagttcct attctctaga aagtatagga acttcagagc
      721 gcttttgaag ctcacgctgc cgcaagcact cagggcgcaa gggctgctaa aggaagcgga
      781 acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg
      841 ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta
      901 catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg
      961 gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc
     1021 caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga ggatcgtttc
     1081 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     1141 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     1201 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     1261 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     1321 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     1381 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     1441 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     1501 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     1561 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     1621 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     1681 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     1741 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     1801 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     1861 ttgacgagtt cttctaataa ggggatcttg aagttcctat tccgaagttc ctattctcta
     1921 gaaagtatag gaacttcgaa gcagctccag cctacacaat cgtgtactgg cgacatggtg
     1981 aactggtctc aagacgtgta atgctgcaat ctgcatgcaa gcttatcgat accgtcgacc
     2041 tcgagggggg gcccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     2101 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     2161 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     2221 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2281 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2341 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2401 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2461 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2521 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2581 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2641 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2701 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2761 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2821 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2881 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2941 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3001 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt tgatcagaaa
     3061 aaaaggatca tatcgtcaat tattacctcc acggggagag cctgagcaaa ctggcctcag
     3121 gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa
     3181 tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc gaatttgctt
     3241 tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt
     3301 aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact
     3361 gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct
     3421 gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa
     3481 cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc
     3541 agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt
     3601 tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt
     3661 ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag
     3721 ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgt aattccggat
     3781 gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt
     3841 tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt
     3901 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg
     3961 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgaca
     4021 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     4081 cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag
     4141 ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggtcg
     4201 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4261 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4321 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4381 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4441 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4501 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4561 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4621 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4681 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaa
//
