LOCUS       sequence-245010-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AM"" 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_AM
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     385..402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    519..540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(555..560,561..578,579..585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    593..609
                     /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     598..620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     617..633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     617..633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     652..672
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        654..672
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    742..775
                     /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             1143..1937
                     /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(1197..1216)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1807..1826
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1950..1997
                     /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(2087..2103)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2088..2104)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2128..2147)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2129..2147)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2157..2174)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2157..2173)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2166..2188)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2315..2770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2402..2421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2612..2633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2796..2900
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3389..4048)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3963..3982
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4049..4151)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      join(4371..4728,1..231)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
       61 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      421 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      481 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      541 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      601 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      661 cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagcccgggc
      721 ggatccgtcg acctgcagtt cgaagttcct attctctaga aagtatagga acttcagagc
      781 gcttttgaag ctcacgctgc cgcaagcact cagggcgcaa gggctgctaa aggaagcgga
      841 acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg
      901 ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta
      961 catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg
     1021 gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc
     1081 caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga ggatcgtttc
     1141 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     1201 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     1261 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     1321 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     1381 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     1441 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     1501 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     1561 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     1621 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     1681 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     1741 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     1801 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     1861 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     1921 ttgacgagtt cttctaataa ggggatcttg aagttcctat tccgaagttc ctattctcta
     1981 gaaagtatag gaacttcgaa gcagctccag cctacacaat cgggcactag ctctaccaca
     2041 cagtgttctc aagacgtgta atgctgcaat ctgcatgcaa gcttatcgat accgtcgacc
     2101 tcgagggggg gcccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     2161 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     2221 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     2281 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2341 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2401 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2461 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2521 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2581 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2641 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2701 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2761 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2821 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2881 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2941 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3001 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3061 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt tgatcagaaa
     3121 aaaaggatca tatcgtcaat tattacctcc acggggagag cctgagcaaa ctggcctcag
     3181 gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa
     3241 tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc gaatttgctt
     3301 tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt
     3361 aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact
     3421 gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct
     3481 gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa
     3541 cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc
     3601 agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt
     3661 tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt
     3721 ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag
     3781 ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgt aattccggat
     3841 gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt
     3901 tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt
     3961 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg
     4021 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgaca
     4081 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     4141 cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag
     4201 ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggtcg
     4261 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4321 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4381 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4441 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4501 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4561 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4621 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4681 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacg
//
