LOCUS       sequence-245008-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKAP plasmid containing the unique DNA barcode ""AK"" 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_AK
FEATURES             Location/Qualifiers
     source          1..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    86..107
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(122..127,128..145,146..152)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    160..176
                     /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     165..187
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     184..200
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     184..200
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     219..239
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        221..239
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    309..342
                     /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             710..1504
                     /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(764..783)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1374..1393
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1517..1564
                     /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(1654..1670)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1655..1671)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1695..1714)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1696..1714)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1724..1741)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1724..1740)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1733..1755)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1882..2337
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1969..1988)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2179..2200
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2363..2467
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2956..3615)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3530..3549
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3616..3718)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3938..4526
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4427..4446
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4680..4697
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
       61 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
      121 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
      181 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
      241 aacaaaagct ggagctccac cgcggtggcg gccgctctag cccgggcgga tccgtcgacc
      301 tgcagttcga agttcctatt ctctagaaag tataggaact tcagagcgct tttgaagctc
      361 acgctgccgc aagcactcag ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc
      421 cagtccgcag aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag
      481 ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct
      541 agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg
      601 taaggttggg aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg
      661 gcgcagggga tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
      721 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
      781 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
      841 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
      901 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
      961 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     1021 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     1081 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     1141 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     1201 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     1261 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     1321 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     1381 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     1441 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     1501 ctaataaggg gatcttgaag ttcctattcc gaagttccta ttctctagaa agtataggaa
     1561 cttcgaagca gctccagcct acacaatcgt cctcacaacg caagcactac ctgtctcaag
     1621 acgtgtaatg ctgcaatctg catgcaagct tatcgatacc gtcgacctcg agggggggcc
     1681 cggtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     1741 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     1801 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     1861 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1921 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1981 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2041 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2101 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2161 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2221 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2281 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     2341 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2401 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2461 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2521 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     2581 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     2641 tcgccccgaa gaacgttttc caatgatgag cactttttga tcagaaaaaa aggatcatat
     2701 cgtcaattat tacctccacg gggagagcct gagcaaactg gcctcaggca tttgagaagc
     2761 acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg
     2821 ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc
     2881 attcatccgc ttattatcac ttattcaggc gtagcaacca ggcgtttaag ggcaccaata
     2941 actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt
     3001 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg
     3061 catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa
     3121 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga
     3181 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca
     3241 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca
     3301 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
     3361 ccatatcacc agctcaccgt ctttcattgc catacgtaat tccggatgag cattcatcag
     3421 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt
     3481 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg
     3541 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt
     3601 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgacaact caaaaaatac
     3661 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     3721 gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt
     3781 tatttattct gcgaagtgat cttccgtcac aggtatttat tcggtcgaaa aggatctagg
     3841 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3901 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3961 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4021 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4081 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4141 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4201 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4261 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4321 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4381 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4441 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4501 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4561 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4621 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4681 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgc
//
