LOCUS       sequence-245003-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AF"" 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_AF
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      40..628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     529..548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     782..799
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    916..937
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(952..957,958..975,976..982)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    990..1006
                     /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     995..1017
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1014..1030
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1014..1030
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1049..1069
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1051..1069
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    1139..1172
                     /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             1540..2334
                     /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(1594..1613)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2204..2223
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    2347..2394
                     /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(2484..2500)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2485..2501)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2525..2544)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2526..2544)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2554..2571)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2554..2570)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2563..2585)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2712..3167
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2799..2818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3009..3030
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3193..3297
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3786..4445)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4360..4379
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4446..4548)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
       61 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
      121 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
      181 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
      241 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
      301 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
      361 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
      421 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
      481 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
      541 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
      601 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
      661 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
      721 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
      781 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
      841 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
      901 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
      961 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     1021 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     1081 ggagctccac cgcggtggcg gccgctctag cccgggcgga tccgtcgacc tgcagttcga
     1141 agttcctatt ctctagaaag tataggaact tcagagcgct tttgaagctc acgctgccgc
     1201 aagcactcag ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag
     1261 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca
     1321 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg
     1381 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg
     1441 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga
     1501 tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     1561 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     1621 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     1681 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     1741 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     1801 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     1861 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     1921 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     1981 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     2041 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     2101 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     2161 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     2221 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     2281 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctaataaggg
     2341 gatcttgaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttcgaagca
     2401 gctccagcct acacaatcgg ctacattgac gtgtccgacc tcaactcaag acgtgtaatg
     2461 ctgcaatctg catgcaagct tatcgatacc gtcgacctcg agggggggcc cggtacccaa
     2521 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     2581 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     2641 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     2701 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2761 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2821 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2881 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2941 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3001 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3061 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3121 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     3181 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3241 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3301 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3361 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3421 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3481 gaacgttttc caatgatgag cactttttga tcagaaaaaa aggatcatat cgtcaattat
     3541 tacctccacg gggagagcct gagcaaactg gcctcaggca tttgagaagc acacggtcac
     3601 actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg
     3661 accctgccct gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc attcatccgc
     3721 ttattatcac ttattcaggc gtagcaacca ggcgtttaag ggcaccaata actgccttaa
     3781 aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
     3841 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
     3901 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata
     3961 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac
     4021 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
     4081 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
     4141 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
     4201 agctcaccgt ctttcattgc catacgtaat tccggatgag cattcatcag gcgggcaaga
     4261 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
     4321 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca
     4381 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
     4441 tccattttag cttccttagc tcctgaaaat ctcgacaact caaaaaatac gcccggtagt
     4501 gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac gtctcatttt
     4561 cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt tatttattct
     4621 gcgaagtgat cttccgtcac aggtatttat tcggtcgaaa aggatctagg tgaagatcct
     4681 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttcca
//
