LOCUS       sequence-210549-        8061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of phage Che9c RecT for oligo-mediated recombineering in 
            mycobacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8061)
  AUTHORS   Murphy KC, Papavinasasundaram K, Sassetti CM
  TITLE     Mycobacterial recombineering.
  JOURNAL   Methods Mol Biol. 2015;1285:177-99. doi: 
            10.1007/978-1-4939-2450-9_10.
  PUBMED    25779316
REFERENCE   2  (bases 1 to 8061)
  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: "Methods Mol
            Biol."; date: "2015"; volume: "1285:177-99. doi"; pages: " 
            10.1007/978-1-4939-2450-9_10"
COMMENT     Sequence Label: pKM402
FEATURES             Location/Qualifiers
     source          1..8061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      20..47
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      1532..1578
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1728..1747)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1739..2554
                     /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(1812..1831)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(2615..2631)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2616..2632)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     CDS             5661..6284
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(5734..5753)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        6314..6416
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             6417..7076
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6483..6502)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      7232..7820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7721..7740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7974..7991
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 actagttgat tagctaagca gaaggccatc ctgacggatg gcctttttgc gtttaatact
       61 gtttaaactc tagaaatatt ggatcgtcgg caccgtcacg gccgtgggag gcggcacgat
      121 ccgcgacgtg atgatcggcc gcatccccac ggtgctgcgc agtgagctct acgccatccc
      181 ggcgttgatc tgtgcgttcg cacgcacagg cccggtgtga gaagggtctc tgacgagcgg
      241 gagaactccc tatcagtgat agagtttgtc ctccctatca gtgatagata ggctctggga
      301 gtacccgtgt gtacgaccag cacggcatac atcatttcga cgccgagaga ttcgccgccc
      361 gaaatgagca cgatccgcat gcttaattaa gaaggagata tacatatggc tgaaaatgct
      421 gtcaccaagc aggattcgcc caaggcaccc gagacgatct cgcaagtgct gcaggtgctc
      481 gtgccgcagc tggcgcgtgc agtgcccaag ggtatggacc ccgaccgcat cgcgcggatc
      541 gtgcaaaccg agatccgcaa gtcgcgcaac gcgaaagctg ctggcatcgc taagcagtcc
      601 ctcgacgact gcacgcaaga gtcatttgcc ggtgcgctgc tgacctcggc cgcgctcggt
      661 ctcgagcccg gtgtcaacgg cgagtgctac ctcgtgccct accgcgacac ccggcgcggc
      721 gtggtcgagt gccagctgat catcggctac cagggcatcg tcaaactgtt ctggcagcac
      781 ccgcgcgcct cccggatcga cgcgcagtgg gtcggcgcga acgacgaatt ccattacacc
      841 atgggcctca atccgacgct gaaacacgtg aaggccaagg gtgatcgggg taatccggtc
      901 tacttctacg cgatcgtcga ggtgaccggc gctgagccgc tgtgggacgt gttcaccgcc
      961 gacgagatca gggaattgcg tcgcggcaag gtcggatcct cgggcgacat caaggacccg
     1021 cagcgctgga tggagcggaa gaccgcgctc aaacaggtgc tgaagctggc accgaagacg
     1081 acgcggctcg acgcggcgat ccgcgccgac gatcgcccgg gcaccgacct gtcacagtcg
     1141 caggcgctcg cgctgccgtc gaccgtcaag ccgacggccg actacatcga cggcgagatc
     1201 gccgagccgc acgaggtcga tacgccaccg aagtcgtcgc gcgcacaacg cgcgcagcgc
     1261 gccaccgcgc cggcgcccga cgtgcagatg gccaatcccg atcagctgaa gcgcctcggc
     1321 gagatccaga aggccgaaaa gtacaacgac gccgattggt tcaagttcct cgccgattcg
     1381 gccggcgtca aagccacgcg cgccgccgac ctcacgttcg acgaggcgaa ggctgtcatc
     1441 gacatgttcg acgggcccaa cgcatgagcg ccccggcgaa ttccgacgcg gtggttgatc
     1501 tgcaactagc gtacgatcga ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     1561 ctgggccttt cgttttatgc catcatggcc gcggtgatca gctagccacc tgacgtcggg
     1621 ggggggggaa agccacgttg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     1681 tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     1741 gagccatatt caacgggaaa cgtcttgctc gaggccgcga ttaaattcca acatggatgc
     1801 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     1861 tcgcttgtat gggaagcccc atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     1921 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     1981 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     2041 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     2101 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     2161 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     2221 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     2281 aatgcataat cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     2341 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     2401 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     2461 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     2521 gcagtttcat ttgatgctcg atgagttttt ctaatcagat atcaagcttt agctaattaa
     2581 ttggggaccc tagaggtccc cttttttatt ttatcgatac cgtcgacctc gagggggggc
     2641 ccccggggat catcgagccg agaacgttat cgaagttggt catgtgtaat cccctcgttt
     2701 gaactttgga ttaagcgtag atacaccctt ggacaagcca gttggattcg gagacaagca
     2761 aattcagcct taaaaagggc gaggccctgc ggtggtggaa caccgcaggg cctctaaccg
     2821 ctcgacgcgc tgcaccaacc agcccgcgaa cggctggcag ccagcgtaag gcgcggctca
     2881 tcgggcggcg ttcgccacga tgtcctgcac ttcgagccaa gcctcgaaca cctgctggtg
     2941 tgcacgactc acccggttgt tgacaccgcg cgcggccgtg cgggctcggt ggggcggctc
     3001 tgtcgccctt gccagcgtga gtagcgcgta cctcacctcg cccaacaggt cgcacacagc
     3061 cgattcgtac gccataaagc caggtgagcc caccagctcc gtaagttcgg gcgctgtgtg
     3121 gctcgtaccc gcgcattcag gcggcagggg gtctaacggg tctaaggcgg cgtgtacggc
     3181 cgccacagcg gctctcagcg gcccggaaac gtcctcgaaa cgacgcatgt gttcctcctg
     3241 gttggtacag gtggttgggg gtgctcggct gtcgctggtg ttccaccacc agggctcgac
     3301 gggagagcgg gggagtgtgc agttgtgggg tggcccctca gcgaaatatc tgacttggag
     3361 ctcgtgtcgg accatacacc ggtgattaat cgtggtctac taccaagcgt gagccacgtc
     3421 gccgacgaat ttgagcagct ctggctgccg tactggccgc tggcaagcga cgatctgctc
     3481 gaggggatct accgccaaag ccgcgcgtcg gccctaggcc gccggtacat cgaggcgaac
     3541 ccaacagcgc tggcaaacct gctggtcgtg gacgtagacc atccagacgc agcgctccga
     3601 gcgctcagcg cccgggggtc ccatccgctg cccaacgcga tcgtgggcaa tcgcgccaac
     3661 ggccacgcac acgcagtgtg ggcactcaac gcccctgttc cacgcaccga atacgcgcgg
     3721 cgtaagccgc tcgcatacat ggcggcgtgc gccgaaggcc ttcggcgcgc cgtcgacggc
     3781 gaccgcagtt actcaggcct catgaccaaa aaccccggcc acatcgcctg ggaaacggaa
     3841 tggctccact cagatctcta cacactcagc cacatcgagg ccgagctcgg cgcgaacatg
     3901 ccaccgccgc gctggcgtca gcagaccacg tacaaagcgg ctccgacgcc gctagggcgg
     3961 aattgcgcac tgttcgattc cgtcaggttg tgggcctatc gtcccgccct catgcggatc
     4021 tacctgccga cccggaacgt ggacggactc ggccgcgcga tctatgccga gtgccacgcg
     4081 cgaaacgccg aattcccgtg caacgacgtg tgtcccggac cgctaccgga cagcgaggtc
     4141 cgcgccatcg ccaacagcat ttggcgttgg atcacaacca agtcgcgcat ttgggcggac
     4201 gggatcgtgg tctacgaggc cacactcagt gcgcgccagt cggccatctc gcggaagggc
     4261 gcagcagcgc gcacggcggc gagcacagtt gcgcggcgcg caaagtccgc gtcagccatg
     4321 gaggcattgc tatgagcgac ggctacagcg acggctacaa ccggcagccg actgtccgca
     4381 aaaagcggcg cgtgaccgcc gccgaaggcg ctcgaatcac cggactatcc gaacgccacg
     4441 tcgtccggct cgtggcgcag gaacgcagcg agtggctcgc cgagcaggct gcacgccgcg
     4501 aacgcatccg cgcctatcac gacgacgagg gccactcttg gccgcaaacg gccaaacatt
     4561 tcgggctgca tctggacacc gttaagcgac tcggctatcg ggcgaggaaa gagcgtgcgg
     4621 cagaacagga agcggctcaa aaggcccaca acgaagccga caatccaccg ctgttctaac
     4681 gcaattgggg agcgggtgtc gcgggggttc cgtggggggt tccgttgcaa cgggtcggac
     4741 aggtaaaagt cctggtagac gctagttttc tggtttgggc catgcctgtc tcgttgcgtg
     4801 tttcgttgcg cccgttttga ataccagcca gacgagacgg ggttctacga atcttggtcg
     4861 ataccaagcc atttccgctg aatatcgggg agctcaccgc cagaatcggt ggttgtggtg
     4921 atgtacgtgg cgaactccgt tgtagtgcct gtggtggcat ccgtggccac tctcgttgca
     4981 cggttcgttg tgccgttaca ggccccgttg acagctcacc gaacgtagtt aaaacatgct
     5041 ggtcaaacta ggtttaccaa cgatacgagt cagctcatct agggccagtt ctaggcgttg
     5101 ttcgttgcgc ggttcgttgc gcatgtttcg tgtggttgct agatggctcc gcaaccacac
     5161 gcttcgaggt tgagtgcttc cagcacgggc gcgatccaga agaacttcgt cgtgcgactg
     5221 tcctcgttga tccttgccga gctgggatgg aagcccggcc gacccaccct ggaggagatg
     5281 atcgaggatg ccagggcctt tcacgcccgc cgctgctgag cgtccgccgc cgggcccgca
     5341 ccgccgtcgg ccggcccgct ccgggctcgc agcagcgggc ttcggcgcgg gcccggggct
     5401 cccgggccgc cgggcggggc tccgcccggc ggccgctgat tagctaagca gaaggccatc
     5461 ctgacggatg cctttttcgc tttaatactg catgcactct agaaatattg gatcgtcgca
     5521 ccgggttaag cctggagtgc ggtggtgcct ggtcggcatt ttcgcagtcg agggctctcg
     5581 tgtagcctgg gcgagttgcc gacgcaggcg accctcctgc cacggatccg catggtacca
     5641 tcaggaggaa tcacttcgca atgtctagat tagataaaag taaagtgatt aacagcgcat
     5701 tagagctgct taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc
     5761 ttggtgtaga gcagcctaca ttgtattggc atgtaaaaaa taagcgggcc ctgctcgacg
     5821 ccttagccat tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa
     5881 gctggcaaga ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc
     5941 gcgatggagc aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg
     6001 aaaatcaatt agccttttta tgccaacaag gtttttcact agagaacgcg ttatatgcac
     6061 tcagcgctgt ggggcatttt accttaggtt gcgtattgga agatcaagag catcaagtcg
     6121 ctaaagaaga aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta
     6181 tcgaattatt tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca
     6241 tctgcggatt agaaaaacaa cttaaatgtg aaagtgggtc ttaatgacca tgggctagat
     6301 ttaaatgaga cgttgatcgg cacgtaagag gttccaactt tcaccataat gaaataagat
     6361 cactaccggg cgtatttttt gagtcatcga gattttcagg agctaaggaa gctaaaatgg
     6421 agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt
     6481 ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta
     6541 cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca
     6601 ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc
     6661 tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt
     6721 tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatactcgc
     6781 aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata
     6841 tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca
     6901 atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca
     6961 aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg
     7021 gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaatttt
     7081 tttaaggcag ttattggtgc ccttaaacgc ctggttgcta cgcctgaata agtgataata
     7141 agcggatgaa tggcagaaat tcgaaagcaa attcgacccg tcatttaaat cactgagcgt
     7201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     7261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     7321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     7381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     7921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     7981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     8041 cgattcatta atgcagctag a
//
