LOCUS       sequence-244098-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AH"" 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_AH
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     128..147
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     381..398
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    515..536
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(551..556,557..574,575..581)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    589..605
                     /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     594..616
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     613..629
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     613..629
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     648..668
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        650..668
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    738..771
                     /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             1139..1933
                     /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(1193..1212)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1803..1822
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1946..1993
                     /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(2083..2099)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2084..2100)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2124..2143)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2125..2143)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2153..2170)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2153..2169)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2162..2184)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2311..2766
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2398..2417)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2608..2629
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2792..2896
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3385..4044)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3959..3978
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4045..4147)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      join(4367..4728,1..227)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
       61 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
      121 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
      181 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
      241 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
      301 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
      361 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
      421 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
      481 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      541 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      601 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      661 actaaaggga acaaaagctg gagctccacc gcggtggcgg ccgctctagc ccgggcggat
      721 ccgtcgacct gcagttcgaa gttcctattc tctagaaagt ataggaactt cagagcgctt
      781 ttgaagctca cgctgccgca agcactcagg gcgcaagggc tgctaaagga agcggaacac
      841 gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat
      901 ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg
      961 gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc
     1021 gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag
     1081 gatctgatgg cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat
     1141 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     1201 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     1261 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     1321 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     1381 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     1441 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     1501 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     1561 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     1621 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     1681 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     1741 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     1801 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     1861 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     1921 cgagttcttc taataagggg atcttgaagt tcctattccg aagttcctat tctctagaaa
     1981 gtataggaac ttcgaagcag ctccagccta cacaatcggg tacaatcacg actcggtggt
     2041 catctcaaga cgtgtaatgc tgcaatctgc atgcaagctt atcgataccg tcgacctcga
     2101 gggggggccc ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     2161 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2221 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     2281 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     2341 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2401 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2461 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2521 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2581 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2641 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2701 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     2761 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2821 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     2881 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     2941 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3001 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3061 tgagagtttt cgccccgaag aacgttttcc aatgatgagc actttttgat cagaaaaaaa
     3121 ggatcatatc gtcaattatt acctccacgg ggagagcctg agcaaactgg cctcaggcat
     3181 ttgagaagca cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga
     3241 cataagcggc tatttaacga ccctgccctg aaccgacgac cgggtcgaat ttgctttcga
     3301 atttctgcca ttcatccgct tattatcact tattcaggcg tagcaaccag gcgtttaagg
     3361 gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtactgttg
     3421 taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat
     3481 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
     3541 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     3601 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     3661 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     3721 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     3781 aacactatcc catatcacca gctcaccgtc tttcattgcc atacgtaatt ccggatgagc
     3841 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     3901 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     3961 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     4021 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgacaactc
     4081 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
     4141 ccgatcaacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac
     4201 accaggattt atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggtcgaaaa
     4261 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4321 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4381 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4441 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4501 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4561 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4621 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4681 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacct
//
