LOCUS       sequence-244097-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AG"" 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_AG
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     123..142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     376..393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    510..531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(546..551,552..569,570..576)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    584..600
                     /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     589..611
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     608..624
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     608..624
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     643..663
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        645..663
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    733..766
                     /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             1134..1928
                     /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(1188..1207)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1798..1817
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1941..1988
                     /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(2078..2094)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2079..2095)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2119..2138)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2120..2138)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2148..2165)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2148..2164)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2157..2179)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2306..2761
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2393..2412)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2603..2624
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2787..2891
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3380..4039)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3954..3973
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4040..4142)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      join(4362..4728,1..222)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
       61 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      361 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      421 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      481 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      541 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      601 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
      661 agggaacaaa agctggagct ccaccgcggt ggcggccgct ctagcccggg cggatccgtc
      721 gacctgcagt tcgaagttcc tattctctag aaagtatagg aacttcagag cgcttttgaa
      781 gctcacgctg ccgcaagcac tcagggcgca agggctgcta aaggaagcgg aacacgtaga
      841 aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga
      901 caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat
      961 agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct
     1021 ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct
     1081 gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     1141 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     1201 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     1261 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     1321 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     1381 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     1441 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     1501 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     1561 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     1621 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     1681 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     1741 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     1801 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     1861 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     1921 tcttctaata aggggatctt gaagttccta ttccgaagtt cctattctct agaaagtata
     1981 ggaacttcga agcagctcca gcctacacaa tcgtcacatg ctcctccagg acactactct
     2041 caagacgtgt aatgctgcaa tctgcatgca agcttatcga taccgtcgac ctcgaggggg
     2101 ggcccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     2161 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2221 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2281 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2341 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2401 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2461 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2521 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2581 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2641 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2701 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     2761 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2821 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2881 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2941 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3001 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3061 gttttcgccc cgaagaacgt tttccaatga tgagcacttt ttgatcagaa aaaaaggatc
     3121 atatcgtcaa ttattacctc cacggggaga gcctgagcaa actggcctca ggcatttgag
     3181 aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa
     3241 gcggctattt aacgaccctg ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc
     3301 tgccattcat ccgcttatta tcacttattc aggcgtagca accaggcgtt taagggcacc
     3361 aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt
     3421 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca
     3481 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga
     3541 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg
     3601 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt
     3661 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac
     3721 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac
     3781 tatcccatat caccagctca ccgtctttca ttgccatacg taattccgga tgagcattca
     3841 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg
     3901 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg
     3961 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc
     4021 cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgac aactcaaaaa
     4081 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat
     4141 caacgtctca ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag
     4201 gatttattta ttctgcgaag tgatcttccg tcacaggtat ttattcggtc gaaaaggatc
     4261 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4501 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacacc
//
