LOCUS       sequence-244950-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AA"" 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_AA
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     156..175
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     409..426
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    543..564
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(579..584,585..602,603..609)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    617..633
                     /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     622..644
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     641..657
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     641..657
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     676..696
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        678..696
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    766..799
                     /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             1167..1961
                     /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(1221..1240)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1831..1850
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1974..2021
                     /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(2111..2127)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2112..2128)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2152..2171)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2153..2171)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2181..2198)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2181..2197)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2190..2212)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2339..2794
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2426..2445)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2636..2657
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2820..2924
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3413..4072)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3987..4006
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4073..4175)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      join(4395..4728,1..255)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
       61 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
      121 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
      181 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
      241 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
      301 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
      361 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      421 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
      481 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      541 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      601 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      661 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctccaccgc
      721 ggtggcggcc gctctagccc gggcggatcc gtcgacctgc agttcgaagt tcctattctc
      781 tagaaagtat aggaacttca gagcgctttt gaagctcacg ctgccgcaag cactcagggc
      841 gcaagggctg ctaaaggaag cggaacacgt agaaagccag tccgcagaaa cggtgctgac
      901 cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
      961 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     1021 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     1081 taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agatctgatc
     1141 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     1201 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     1261 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     1321 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     1381 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     1441 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     1501 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     1561 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     1621 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     1681 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     1741 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     1801 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     1861 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     1921 agcgcatcgc cttctatcgc cttcttgacg agttcttcta ataaggggat cttgaagttc
     1981 ctattccgaa gttcctattc tctagaaagt ataggaactt cgaagcagct ccagcctaca
     2041 caatcgactc aagcaccagc aggagacttc tctcaagacg tgtaatgctg caatctgcat
     2101 gcaagcttat cgataccgtc gacctcgagg gggggcccgg tacccaattc gccctatagt
     2161 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2221 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2281 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     2341 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2401 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2461 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2521 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2581 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2641 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2701 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2761 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     2821 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2881 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2941 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3001 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3061 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3121 tgatgagcac tttttgatca gaaaaaaagg atcatatcgt caattattac ctccacgggg
     3181 agagcctgag caaactggcc tcaggcattt gagaagcaca cggtcacact gcttccggta
     3241 gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc ctgccctgaa
     3301 ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt catccgctta ttatcactta
     3361 ttcaggcgta gcaaccaggc gtttaagggc accaataact gccttaaaaa aattacgccc
     3421 cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc
     3481 catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg
     3541 tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta
     3601 aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa
     3661 accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt
     3721 gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt
     3781 gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt
     3841 tcattgccat acgtaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg
     3901 ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct
     3961 gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac
     4021 gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt
     4081 ccttagctcc tgaaaatctc gacaactcaa aaaatacgcc cggtagtgat cttatttcat
     4141 tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg
     4201 cccagggctt cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt
     4261 ccgtcacagg tatttattcg gtcgaaaagg atctaggtga agatcctttt tgataatctc
     4321 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4381 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4441 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4501 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4561 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4621 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4681 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgc
//
