LOCUS       sequence-69379-e        5096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for ectopic constitutive expression of two mycobacterial proteins in
            one vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5096)
  AUTHORS   Parikh A, Kumar D, Chawla Y, Kurthkoti K, Khan S, Varshney U, 
            Nandicoori VK
  TITLE     Development of a new generation of vectors for gene expression, gene
            replacement, and protein-protein interaction studies in 
            mycobacteria.
  JOURNAL   Appl Environ Microbiol. 2013 Mar;79(5):1718-29. doi: 
            10.1128/AEM.03695-12. Epub 2013 Jan 11.
  PUBMED    23315736
REFERENCE   2  (bases 1 to 5096)
  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"; doi: 
            "10.1128/AEM.03695-12"; journalName: "Appl Environ Microbiol"; date:
            "2013-03"; volume: "79"; issue: "5"; pages: "1718-29"
COMMENT     Sequence Label: pST-2K
FEATURES             Location/Qualifiers
     source          1..5096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(26..125)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             470..487
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      complement(517..560)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             956..979
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      complement(998..1040)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1069..1115
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1265..1284)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1276..2091
                     /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     complement(1349..1368)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2423..3011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2912..2931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agtactagta agggtgggcg cgccgaccca gctttcttgt acaaagttgg cattataaga
       61 aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt
      121 atttgccatc cagctgatac taatattgga tcgtcggcac cgtcacggcc gtgggaggcg
      181 gcacgatccg cgacgtgatg atcggccgca tccccacggt gctgcgcagt gagctctacg
      241 ccatcccggc gttgatctgt gcgttcgcac gcacaggccc ggtgtgagaa gggtctctga
      301 cgagcgggag aactccctat cagtgataga gtttgtcctc cctatcagtg atagataggc
      361 tctgggagta cccgtgtgta cgaccagcac ggcatacatc atttcgacgc cgagagattc
      421 gccgcccgaa atgagcacga tccgcatgcg gaggaatcac ttcgcaatgc accaccacca
      481 ccaccatatg ggatccgata tctctagagc ggccgcataa aacgaaaggc tcagtcgaaa
      541 gactgggcct ttcgttttat agggccgccc ccttcaccca gctgagtact aatattggat
      601 cgtcggcacc gtcacggccg tgggaggcgg cacgatccgc gacgtgatga tcggccgcat
      661 ccccacggtg ctgcgcagtg agctctacgc catcccggcg ttgatctgtg cgttcgcacg
      721 cacaggcccg gtgtgagaag ggtctctgac gagcgggaga acccacccgg ggtgggcgag
      781 tttgtcctgc gtgtgctcgg tcgagtaggc tctgggagta cccgtgtgta cgaccagcac
      841 ggcatacatc atttcgacgc cgagagattc gccgcccgaa atgagcacga tccgcatgcg
      901 ggcccggagg aatcacttcg caatggaatt cctgcagcac gtgttaatta agcttgacta
      961 caaggacgac gacgacaagt aaaagctatc agttaactaa aacgaaaggc tcagtcgaaa
     1021 gactgggcct ttcgttttat aactagcgta cgatcgactg ccaggcatca aataaaacga
     1081 aaggctcagt cgaaagactg ggcctttcgt tttatgccat catggccgcg gtgatcagct
     1141 agccacctga cgtcgggggg gggggaaagc cacgttgtgt ctcaaaatct ctgatgttac
     1201 attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt
     1261 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctcgag gccgcgatta
     1321 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     1381 tcaggtgcga caatctatcg cttgtatggg aagccccatg cgccagagtt gtttctgaaa
     1441 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     1501 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     1561 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     1621 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     1681 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     1741 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     1801 gaacaagtct ggaaagaaat gcataatctt ttgccattct caccggattc agtcgtcact
     1861 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     1921 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     1981 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     2041 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg
     2101 gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg cggctttgtt
     2161 gaataaatcg aacttttgct gagttgaagg atcagatcac gcatcttccc gacaacgcag
     2221 accgttccgt ggcaaagcaa aagttcaaaa tcaccaactg gtccacctac aacaaagctc
     2281 tcatcaaccg tggctccctc actttctggc tggatgatgg ggcgattcag gcctggtatg
     2341 agtcagcaac accttcttca cgaggcagac ctcactagtt ccactgagcg tcagaccccg
     2401 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2461 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2521 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2581 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2641 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2701 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2761 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagcattgag
     2821 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2881 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2941 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3001 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3061 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3121 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcaacgcg ttacgtagag
     3181 cccaccagct ccgtaagttc gggtgctgtg tggctcgtac ccgcgcattc aggcggcagg
     3241 gggtctaacg ggtctaaggc ggcgtgtacg gccgccacag cggctcttag cggcccggaa
     3301 acgtcctcga aacgacgcat gtgttcctcc tggttggtac aggtggttgg gggtgctcgg
     3361 ctgtcgctgg tgtttcatca tcagggctcg acgggagagc gggggagtgt gcagttgtgg
     3421 ggtggcccct cagcgaaata tctgacttgg agctcgtgtc ggaccataca ccggtgatta
     3481 atcgtggttt attatcaagc gtgagccacg tcgccgacga atttgagcag ctctggctgc
     3541 cgtactggtc cctggcaagc gacgatctgc tcgaggggat ctaccgccaa agccgcgcgt
     3601 cggccctagg ccgccggtac atcgaggcga acccaacagc gctggcaaac ctgctggtcg
     3661 tggacgtaga ccatccagac gcagcgctcc gagcgctcag cgcccggggg tcccatccgc
     3721 tgcccaacgc gatcgtgggc aatcgcgcca acggccacgc acacgcagtg tgggcactca
     3781 acgcccctgt tccacgcacc gaatacgcgc ggcgtaagcc gctcgcatac atggcggcgt
     3841 gcgccgaagg ccttcggcgc gccgtcgatg gcgaccgcag ttactcaggc ctcatgacca
     3901 aaaaccccgg ccacatcgcc tgggaaacgg aatggctcca ctcagatctc tacacactca
     3961 gccacatcga ggccgagctc ggcgcgaaca tgccaccgcc gcgctggcgt cagcagacca
     4021 cgtacaaagc ggctccgacg ccgctagggc ggaattgcgc actgttcgat tccgtcaggt
     4081 tgtgggccta tcttcccgcc ctcatgcgga tctacctgcc gacccggaac gtggacggac
     4141 tcggccgcgc gatctatgcc gagtgccacg cgcgaaacgc cgaatttccg tgcaacgacg
     4201 tgtgtcccgg accgctaccg gacagcgagg tccgcgccat cgccaacagc atttggcgtt
     4261 ggatcacaac caagtcgcgc atttgggcgg acgggatcgt ggtctacgag gccacactca
     4321 gtgcgcgcca tgcggccatc tcgcggaagg gcgcagcagc gcgcacggcg gcgagcacag
     4381 ttgcgcggcg cgcaaagtcc gcgtcagcca tggaggcatt gctatgagcg acggctacag
     4441 cgacggctac agcgacggct acaactggca gccgactgtc cgcaaaaagc ggcgcgtgac
     4501 cgccgccgaa ggcgctcgaa tcaccggact atccgaacgc cacgtcgtcc ggctcgtggc
     4561 gcaggaacgc agcgagtggt tcgccgagca ggctgcacgc cgcgaacgca tccgcgccta
     4621 tcacgacgac gagggccact cttggccgca aacggccaaa catttcgggc tgcatctgga
     4681 caccgttaag cgactcggct atcgggcgag gaaagagcgt gcggcagaac aggaagcggc
     4741 tcaaaaggcc cacaacgaag ccgacaatcc accgctgttc taacgcaatt ggggagcggg
     4801 tgtcgcgggg gttccgtggg gggttccgtt gcaacgggtc ggacaggtaa aagtcctggt
     4861 agacgctagt tttctggttt gggccatgcc tgtctcgttg cgtgtttcgt tgcgtccgtt
     4921 ttgaatacca gccagacgag acggggttct acgaatcttg gtcgatacca agccatttcc
     4981 gctgaatatc ggggagctca ccgccagaat cggtggttgt ggtgatgtac gtggcgaact
     5041 ccgttgtagt gcctgtggtg gcatccgtgg cggtaccgcc tgacgccccc cggatc
//
