LOCUS       sequence-239200-        4703 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Designed for the incorporation of DNA barcode sequences for 
            subsequent allelic exchange on bacterial chromosomes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4703)
  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 4703)
  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
FEATURES             Location/Qualifiers
     source          1..4703
                     /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"
     protein_bind    complement(751..798)
                     /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(811..1605)
                     /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(922..941)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1532..1551
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     protein_bind    complement(1973..2006)
                     /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(2076..2096)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2076..2094)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2115..2131)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2115..2131)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2128..2150)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2139..2155
                     /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(2163..2169,2170..2187,2188..2193))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2208..2229
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2346..2363)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2517..3105)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2597..2616)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3325..3427
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3428..4087
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3494..3513)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4575..4680)
                     /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 ttgagcgatt gtgtaggctg gagctgcttc gaagttccta tactttctag agaataggaa
      781 cttcggaata ggaacttcaa gatcccctta ttagaagaac tcgtcaagaa ggcgatagaa
      841 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
      901 ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc
      961 cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat
     1021 attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc
     1081 cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
     1141 ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
     1201 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat
     1261 gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc
     1321 gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg
     1381 aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc
     1441 accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
     1501 ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac
     1561 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca
     1621 tcctgtctct tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc
     1681 catccagttt actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc
     1741 cggttcgctt gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca
     1801 agctacctgc tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac
     1861 attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta
     1921 gcagcccttg cgccctgagt gcttgcggca gcgtgagctt caaaagcgct ctgaagttcc
     1981 tatactttct agagaatagg aacttcgaac tgcaggtcga cggatccgcc cgggctagag
     2041 cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
     2101 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     2161 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     2221 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2281 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2341 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2401 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2461 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2521 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2581 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2641 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2701 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2761 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2821 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2881 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2941 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3001 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3061 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3121 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3181 agattatcaa aaaggatctt cacctagatc cttttcgacc gaataaatac ctgtgacgga
     3241 agatcacttc gcagaataaa taaatcctgg tgtccctgtt gataccggga agccctgggc
     3301 caacttttgg cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa
     3361 tgaaataaga tcactaccgg gcgtattttt tgagttgtcg agattttcag gagctaagga
     3421 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
     3481 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
     3541 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
     3601 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattacgta tggcaatgaa
     3661 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca
     3721 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca
     3781 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt
     3841 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt
     3901 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac
     3961 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg
     4021 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg
     4081 ggcgtaattt ttttaaggca gttattggtg cccttaaacg cctggttgct acgcctgaat
     4141 aagtgataat aagcggatga atggcagaaa ttcgaaagca aattcgaccc ggtcgtcggt
     4201 tcagggcagg gtcgttaaat agccgcttat gtctattgct ggtttaccgg tttattgact
     4261 accggaagca gtgtgaccgt gtgcttctca aatgcctgag gccagtttgc tcaggctctc
     4321 cccgtggagg taataattga cgatatgatc ctttttttct gatcaaaagt gctcatcatt
     4381 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4441 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4501 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4561 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca agggttattg
     4621 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4681 cacatttccc cgaaaagtgc cac
//
