LOCUS       sequence-244948-        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"
     primer_bind     116..135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     369..386
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    503..524
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(539..544,545..562,563..569)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    577..593
                     /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     582..604
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     601..617
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     601..617
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     636..656
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        638..656
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    726..759
                     /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             1127..1921
                     /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(1181..1200)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1791..1810
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1934..1981
                     /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(2046..2062)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2047..2063)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2087..2106)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2088..2106)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2116..2133)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2116..2132)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2125..2147)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2274..2729
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2361..2380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2571..2592
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2755..2859
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3348..4007)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3922..3941
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4008..4110)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      join(4330..4703,1..215)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
       61 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      121 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      181 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      241 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      301 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
      361 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
      421 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      481 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      541 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      601 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
      661 aaaagctgga gctccaccgc ggtggcggcc gctctagccc gggcggatcc gtcgacctgc
      721 agttcgaagt tcctattctc tagaaagtat aggaacttca gagcgctttt gaagctcacg
      781 ctgccgcaag cactcagggc gcaagggctg ctaaaggaag cggaacacgt agaaagccag
      841 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga
      901 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga
      961 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa
     1021 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg
     1081 caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     1141 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     1201 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     1261 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     1321 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     1381 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     1441 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     1501 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     1561 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     1621 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     1681 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     1741 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     1801 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     1861 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttcta
     1921 ataaggggat cttgaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     1981 cgaagcagct ccagcctaca caatcgctca agacgtgtaa tgctgcaatc tgcatgcaag
     2041 cttatcgata ccgtcgacct cgaggggggg cccggtaccc aattcgccct atagtgagtc
     2101 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     2161 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     2221 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     2281 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2341 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2401 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2461 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2521 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2581 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2641 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2701 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     2761 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2821 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2881 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2941 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3001 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3061 agcacttttt gatcagaaaa aaaggatcat atcgtcaatt attacctcca cggggagagc
     3121 ctgagcaaac tggcctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa
     3181 taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac
     3241 gaccgggtcg aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag
     3301 gcgtagcaac caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc
     3361 tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
     3421 caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
     3481 tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
     3541 aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
     3601 ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
     3661 aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
     3721 tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
     3781 gccatacgta attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
     3841 taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
     3901 gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
     3961 cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta
     4021 gctcctgaaa atctcgacaa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg
     4081 tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccaaaa gttggcccag
     4141 ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc
     4201 acaggtattt attcggtcga aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4261 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4321 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4381 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4441 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4501 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4561 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4621 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4681 gcgaacgacc tacaccgaac tga
//
