LOCUS       sequence-244994-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AU"" 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_AU
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     369..386
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    503..524
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(539..544,545..562,563..569)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    577..593
                     /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     582..604
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     601..617
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     601..617
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     636..656
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        638..656
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    726..759
                     /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             1127..1921
                     /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(1181..1200)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1791..1810
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1934..1981
                     /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(2071..2087)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2072..2088)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2112..2131)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2113..2131)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2141..2158)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2141..2157)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2150..2172)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2299..2754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2386..2405)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2596..2617
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2780..2884
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3373..4032)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3947..3966
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4033..4135)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      join(4355..4728,1..215)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
       61 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      121 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      181 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      241 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      301 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
      361 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
      421 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      481 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      541 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      601 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
      661 aaaagctgga gctccaccgc ggtggcggcc gctctagccc gggcggatcc gtcgacctgc
      721 agttcgaagt tcctattctc tagaaagtat aggaacttca gagcgctttt gaagctcacg
      781 ctgccgcaag cactcagggc gcaagggctg ctaaaggaag cggaacacgt agaaagccag
      841 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga
      901 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga
      961 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa
     1021 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg
     1081 caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     1141 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     1201 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     1261 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     1321 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     1381 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     1441 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     1501 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     1561 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     1621 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     1681 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     1741 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     1801 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     1861 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttcta
     1921 ataaggggat cttgaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     1981 cgaagcagct ccagcctaca caatcgctct ctggatgact cgcacagtcg tctcaagacg
     2041 tgtaatgctg caatctgcat gcaagcttat cgataccgtc gacctcgagg gggggcccgg
     2101 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     2161 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     2221 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     2281 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2341 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2401 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2461 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2521 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2581 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2641 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2701 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     2761 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2821 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2881 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2941 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3001 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3061 ccccgaagaa cgttttccaa tgatgagcac tttttgatca gaaaaaaagg atcatatcgt
     3121 caattattac ctccacgggg agagcctgag caaactggcc tcaggcattt gagaagcaca
     3181 cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta
     3241 tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt
     3301 catccgctta ttatcactta ttcaggcgta gcaaccaggc gtttaagggc accaataact
     3361 gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag
     3421 cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat
     3481 cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt
     3541 gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac
     3601 gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc
     3661 cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag
     3721 cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca
     3781 tatcaccagc tcaccgtctt tcattgccat acgtaattcc ggatgagcat tcatcaggcg
     3841 ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa
     3901 aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa
     3961 tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat
     4021 ttttttctcc attttagctt ccttagctcc tgaaaatctc gacaactcaa aaaatacgcc
     4081 cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc
     4141 tcattttcgc caaaagttgg cccagggctt cccggtatca acagggacac caggatttat
     4201 ttattctgcg aagtgatctt ccgtcacagg tatttattcg gtcgaaaagg atctaggtga
     4261 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4321 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4381 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4441 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4501 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4561 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4621 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4681 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactga
//
