LOCUS       sequence-73915-e        5991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kanamycin and Chloramphenicol resistant; destination vector 
            engineered to integrate into the mycobacteriophage att-L5 site and 
            designed to be used with the multisite Gateway cloning system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5991)
  AUTHORS   Kim JH, O'Brien KM, Sharma R, Boshoff HI, Rehren G, Chakraborty S, 
            Wallach JB, Monteleone M, Wilson DJ, Aldrich CC, Barry CE 3rd, Rhee 
            KY, Ehrt S, Schnappinger D
  TITLE     A genetic strategy to identify targets for the development of drugs 
            that prevent bacterial persistence.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Nov 4.
  PUBMED    24191058
REFERENCE   2  (bases 1 to 5991)
  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: "Proc Natl 
            Acad Sci U S A. 2013 Nov 4."
COMMENT     Sequence Label: pDE43-MCK
FEATURES             Location/Qualifiers
     source          1..5991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(37..83)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     340..362
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     359..375
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     359..375
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    395..518
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(559..864)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(661..680)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1184..1864)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     1779..1798
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1918..1924,1925..1942,1943..1948))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1973..2096)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     rep_origin      complement(2350..2938)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2430..2449)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3270..4085)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3993..4012
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4077..4096
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tgatcaccgc ggccatgatg gccggacaaa caacagataa aacgaaaggc ccagtctttc
       61 gactgagcct ttcgttttat ttgatgcctg gcagtcgatc gtacgctagt taactacgtc
      121 gacatcgatt taattaatat gcatcaattg atttaaatac tagtgttaac tatttaattg
      181 gggaccctag aggtcccctt ttttatttta aaaatttttt cnaaggggac ctctagggtc
      241 cccagctggc tagcgagtca tgaggtnacc atgttctttc ctgcgttatc ccctgattct
      301 gtggataacc gtattaccgc ctttgagtga gctgatacca gcggataaca atttcacaca
      361 ggaaacagct atgaccatga ttacgccaag ctatcaactt tgtatagaaa agttgaacga
      421 gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact
      481 acataatact gtaaaacaca acatatccag tcactatggt cgacctgcag actggctgtg
      541 tataagggag cctgacattt atattcccca gaacatcagg ttaatggcgt ttttgatgtc
      601 attttcgcgg tggctgagat cagccacttc ttccccgata acggagaccg gcacactggc
      661 catatcggtg gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc
      721 acgggggact ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc
      781 gggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt
      841 ataggtgtaa accttaaact gcatttcacc agcccctgtt ctcgtcggca aaagagccgt
      901 tcatttcaat aaaccgggcg acctcagcca tcccttcctg attttccgct ttccagcgtt
      961 cggcacgcag acgacgggct tcattctgca tggttgtgct taccgaaccg gagatattga
     1021 catcatatat gccttgagca actgatagct gtcgctgtca actgtcactg taatacgctg
     1081 cttcatagca tccctctttt tgacatactt cgggtataca tatcagtata tattcttata
     1141 ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg cttttagtaa gccggatcct
     1201 ctagattacg ccccgcctgc cactcatcgc agtactgttg taattcatta agcattctgc
     1261 cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc atcagcacct
     1321 tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat
     1381 tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca
     1441 tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt
     1501 gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa
     1561 acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca
     1621 gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg cgggcaagaa
     1681 tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg
     1741 taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa
     1801 aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg atttttttct
     1861 ccattttagc ttccttagct cctgaaaatc tcgacggatc ctaactcaaa atccacacat
     1921 tatacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgcggcc gccatagtga
     1981 ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa
     2041 tatattgata tttatatcat tttacgtttc tcgttcaact ttattataca tagttgctta
     2101 agctgcaggg gaccctagag gtcccctttt ttattttaaa aattttttat aaccggtaag
     2161 aagtccaaca cgccgctcct ccccgatgtt gcgtagccgc ttcaagctta agcaacggcc
     2221 ccagtgagta gcgactttgg atccggtacc cgcaacggtc aaccagcgtc tacgcgtttc
     2281 actggccgac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2341 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2401 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2461 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2521 ctcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2581 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2641 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2701 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2761 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     2821 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2881 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2941 agatcctttg atcttttcta cggggtctga cgctcagtgg aactagtgag gtctgcctcg
     3001 tgaagaaggt gttgctgact cataccaggc ctgaatcgcc ccatcatcca gccagaaagt
     3061 gagggagcca cggttgatga gagctttgtt gtaggtggac cagttggtga ttttgaactt
     3121 ttgctttgcc acggaacggt ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc
     3181 agcaaaagtt cgatttattc aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc
     3241 cagtgttaca accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac
     3301 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     3361 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     3421 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3481 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagattatgc
     3541 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     3601 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     3661 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3721 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     3781 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3841 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     3901 gcaacgctac ctttgccatg tttcagaaac aactctggcg catggggctt cccatacaag
     3961 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     4021 tcagcatcca tgttggaatt taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg
     4081 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat
     4141 atatttttat cttgtgcaat gtaacatcag agattttgag acacaacgtc gctttgttgc
     4201 tagctggcgg gaacggcaat gctcctggcg gcggcggggc tggtgcccag gtctccgcac
     4261 agagcggcgg tgctggcgct cgcggccagg cgtggttctt cgcgtactga caagaaaccc
     4321 ccctctttag gactcagtgt ccttgggagg ggggcttttt gcgtttcagg aggtcttggc
     4381 cagcttggac atcgcctcag cgatagcctc gtcgcgggcc tcagacgcca tctggtactt
     4441 catcgccatc ctaggagtcg tgtgaccgag acgggccatc agctccttgg tcgtcgcacc
     4501 tgcctgagcg gcgaacgtag cgccgacagc gcggaggtcg tggatgcgga gttccggccg
     4561 accgatcttg gcgtagccac gcttcagcga cttggtgaac gcggacttcg acagccggtt
     4621 gccctgcgtc gtggtcacca ggaatgcctc ggggcccttg ttcatcttcg tacggtcctt
     4681 catgtgcgct cggatcatct ccgcgacgtg aggcggaacc gtcacaggac gcttcgaccg
     4741 gacggtcttg gcgttgccaa cgacgatctt gttccccacg cgggaagcgc cacggcgcac
     4801 ccggagcttc atcgtcatgc cgtcgtccac gatgtccttg cggcgaagct cgatcagctc
     4861 tccgaaccgg aggctcgtcc acgccaggat gtatgccgcg atccggtagt gctcgaagat
     4921 ctcagcggcg acgatgtcca gctcctcagg cgtcagcgcc tctacgtcgc gctcatcggc
     4981 tgccttctgc tcgatccggc acgggttctc tgcgatcagc ttgtcctcga ccgctgtgtt
     5041 catcaccgcc cggaggacgt tgtaggcatg ccggcgggca gtcgggtgct tcctacccat
     5101 cccggcccac cacgcacgca ccagagctgg cgtcatctct gtgaccgcca cttcacctag
     5161 caccgggtag atgcggcgct ccgcgtgccc gctgtacaga tccctggtgc cgtctgcgag
     5221 gtcgcgctcc acgagccact tccgggtgta ctcctccagc gtgatggcgc tggcggctgc
     5281 cttcttcgcc cggtcctgtg gaggggtcca ggtctccatc tcgatgagcc gcttctcgcc
     5341 cgcgagccag gcttcggcgt ccatcttgtt gtcgtaggtc tgcagcgcgt agtacctcac
     5401 accgtcctgc gggttgacgt atgaggcttg gatcctcccg ctgcgctgag tcttcagcga
     5461 tccccatccg cgacgtgcca actaggtctc ctctcgtcgt gaacaaggct accgggttgc
     5521 aactcctgtg caactctcag gcttcaacgc gcttctacga cctgcaattt ctttccactt
     5581 agaggatgca gccgagaggg ggtaaaaacc tatcttgacc ggcccatata tgtggtcggc
     5641 agacacccat tcttccaaac tagctacgcg ggttcgattc ccgtcgcccg ctccgctggt
     5701 cagagggtgt tttcgccctc tggccatttt tctttccagg ggtctgcaac tcttgtgcga
     5761 ctcttctgac ctgggcatac gcggttgcaa cgcatccctg atctggctac tttcgatgct
     5821 gacaaacgaa tagagccccc cgcctgcgcg aacagacgag gggcattcac accagattgg
     5881 agctggtgca gtgaagagaa tagaccggga caaggttgca ccgggagttg cagcggtcgg
     5941 aaccctcgcc gtcggcgggc tggcgttcgc cctgtcgttc acggctctag c
//
