LOCUS       sequence-239244-        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"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gap             726..750
                     /estimated_length=25
     protein_bind    complement(776..823)
                     /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)."
     CDS             complement(836..1630)
                     /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(947..966)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1557..1576
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     protein_bind    complement(1998..2031)
                     /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)"
     primer_bind     complement(2101..2121)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2101..2119)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2140..2156)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2140..2156)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2153..2175)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2164..2180
                     /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)."
     promoter        complement(join(2188..2194,2195..2212,2213..2218))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2233..2254
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2371..2388)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2542..3130)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2622..2641)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3350..3452
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3453..4112
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3519..3538)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4600..4705)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgcatgc agattgcagc attacacgtc
      721 ttgagnnnnn nnnnnnnnnn nnnnnnnnnn cgattgtgta ggctggagct gcttcgaagt
      781 tcctatactt tctagagaat aggaacttcg gaataggaac ttcaagatcc ccttattaga
      841 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
      901 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
      961 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     1021 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
     1081 gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     1141 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     1201 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     1261 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     1321 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     1381 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
     1441 cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
     1501 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     1561 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     1621 gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatcttgat cccctgcgcc
     1681 atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc ccaaccttac
     1741 cagagggcgc cccagctggc aattccggtt cgcttgctgt ccataaaacc gcccagtcta
     1801 gctatcgcca tgtaagccca ctgcaagcta cctgctttct ctttgcgctt gcgttttccc
     1861 ttgtccagat agcccagtag ctgacattca tccggggtca gcaccgtttc tgcggactgg
     1921 ctttctacgt gttccgcttc ctttagcagc ccttgcgccc tgagtgcttg cggcagcgtg
     1981 agcttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt cgaactgcag
     2041 gtcgacggat ccgcccgggc tagagcggcc gccaccgcgg tggagctcca gcttttgttc
     2101 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     2161 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     2221 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     2281 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     2341 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     2401 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     2461 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     2521 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2581 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2641 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2701 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2761 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2821 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2881 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2941 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     3001 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3061 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3121 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     3181 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     3241 cgaccgaata aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc
     3301 ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg
     3361 taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt
     3421 tgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca
     3481 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc
     3541 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga
     3601 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc
     3661 atccggaatt acgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc
     3721 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc
     3781 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa
     3841 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct
     3901 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg
     3961 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc
     4021 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac
     4081 agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt
     4141 aaacgcctgg ttgctacgcc tgaataagtg ataataagcg gatgaatggc agaaattcga
     4201 aagcaaattc gacccggtcg tcggttcagg gcagggtcgt taaatagccg cttatgtcta
     4261 ttgctggttt accggtttat tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc
     4321 ctgaggccag tttgctcagg ctctccccgt ggaggtaata attgacgata tgatcctttt
     4381 tttctgatca aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4441 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4501 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4561 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4621 ttgaagcatt tatcaagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4681 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
