LOCUS       sequence-245002-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AR"" 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_AR
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     363..382
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     616..633
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    750..771
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(786..791,792..809,810..816)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    824..840
                     /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     829..851
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     848..864
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     848..864
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     883..903
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        885..903
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    973..1006
                     /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             1374..2168
                     /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(1428..1447)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2038..2057
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    2181..2228
                     /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(2318..2334)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2319..2335)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2359..2378)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2360..2378)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2388..2405)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2388..2404)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2397..2419)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2546..3001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2633..2652)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2843..2864
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3027..3131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3620..4279)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4194..4213
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4280..4382)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      join(4602..4728,1..462)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
       61 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      121 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      181 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      241 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      301 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      361 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      421 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      481 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      541 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      601 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      661 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      721 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      781 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      841 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
      901 agggaacaaa agctggagct ccaccgcggt ggcggccgct ctagcccggg cggatccgtc
      961 gacctgcagt tcgaagttcc tattctctag aaagtatagg aacttcagag cgcttttgaa
     1021 gctcacgctg ccgcaagcac tcagggcgca agggctgcta aaggaagcgg aacacgtaga
     1081 aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga
     1141 caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat
     1201 agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct
     1261 ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct
     1321 gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     1381 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     1441 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     1501 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     1561 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     1621 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     1681 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     1741 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     1801 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     1861 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     1921 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     1981 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     2041 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     2101 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     2161 tcttctaata aggggatctt gaagttccta ttccgaagtt cctattctct agaaagtata
     2221 ggaacttcga agcagctcca gcctacacaa tcgcagtgtg ttacacctca gtacgacact
     2281 caagacgtgt aatgctgcaa tctgcatgca agcttatcga taccgtcgac ctcgaggggg
     2341 ggcccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     2401 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2461 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2521 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2581 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2641 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2701 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2761 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2821 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2881 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2941 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     3001 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3061 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt ttgatcagaa aaaaaggatc
     3361 atatcgtcaa ttattacctc cacggggaga gcctgagcaa actggcctca ggcatttgag
     3421 aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa
     3481 gcggctattt aacgaccctg ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc
     3541 tgccattcat ccgcttatta tcacttattc aggcgtagca accaggcgtt taagggcacc
     3601 aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt
     3661 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca
     3721 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga
     3781 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg
     3841 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt
     3901 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac
     3961 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac
     4021 tatcccatat caccagctca ccgtctttca ttgccatacg taattccgga tgagcattca
     4081 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg
     4141 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg
     4201 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc
     4261 cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgac aactcaaaaa
     4321 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat
     4381 caacgtctca ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag
     4441 gatttattta ttctgcgaag tgatcttccg tcacaggtat ttattcggtc gaaaaggatc
     4501 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4561 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4621 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4681 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcaga
//
