LOCUS       sequence-245019-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AS"" 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_AS
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    520..541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(556..561,562..579,580..586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    594..610
                     /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     599..621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     618..634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     618..634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     653..673
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        655..673
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    743..776
                     /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             1144..1938
                     /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(1198..1217)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1808..1827
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1951..1998
                     /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(2088..2104)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2089..2105)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2129..2148)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2130..2148)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2158..2175)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2158..2174)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2167..2189)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2316..2771
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2403..2422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2613..2634
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2797..2901
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3390..4049)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3964..3983
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4050..4152)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      join(4372..4728,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      541 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      601 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
      661 ccctcactaa agggaacaaa agctggagct ccaccgcggt ggcggccgct ctagcccggg
      721 cggatccgtc gacctgcagt tcgaagttcc tattctctag aaagtatagg aacttcagag
      781 cgcttttgaa gctcacgctg ccgcaagcac tcagggcgca agggctgcta aaggaagcgg
      841 aacacgtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg
      901 gctatctgga caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt
      961 acatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct
     1021 ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg
     1081 ccaaggatct gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt
     1141 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     1201 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     1261 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     1321 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     1381 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     1441 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     1501 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     1561 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     1621 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     1681 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     1741 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     1801 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     1861 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     1921 cttgacgagt tcttctaata aggggatctt gaagttccta ttccgaagtt cctattctct
     1981 agaaagtata ggaacttcga agcagctcca gcctacacaa tcgcagagtg taactcctgt
     2041 ctacgagact caagacgtgt aatgctgcaa tctgcatgca agcttatcga taccgtcgac
     2101 ctcgaggggg ggcccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     2161 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     2221 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     2281 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     2341 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2401 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2461 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2521 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2581 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2641 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2701 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2761 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2821 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2881 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2941 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3001 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3061 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt ttgatcagaa
     3121 aaaaaggatc atatcgtcaa ttattacctc cacggggaga gcctgagcaa actggcctca
     3181 ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca
     3241 atagacataa gcggctattt aacgaccctg ccctgaaccg acgaccgggt cgaatttgct
     3301 ttcgaatttc tgccattcat ccgcttatta tcacttattc aggcgtagca accaggcgtt
     3361 taagggcacc aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac
     3421 tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc
     3481 tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa
     3541 acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc
     3601 cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg
     3661 ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg
     3721 tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa
     3781 gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg taattccgga
     3841 tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt
     3901 ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat
     3961 tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg
     4021 gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgac
     4081 aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt
     4141 acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc ggtatcaaca
     4201 gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat ttattcggtc
     4261 gaaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4321 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4381 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4441 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4501 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4561 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4621 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4681 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaac
//
