LOCUS       sequence-292611-        6374 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of Mtb CblA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6374)
  AUTHORS   Ruetz M, Campanello GC, Purchal M, Shen H, McDevitt L, Gouda H, 
            Wakabayashi S, Zhu J, Rubin EJ, Warncke K, Mootha VK, Koutmos M, 
            Banerjee R
  TITLE     Itaconyl-CoA forms a stable biradical in methylmalonyl-CoA mutase 
            and derails its activity and repair.
  JOURNAL   Science. 2019 Nov 1;366(6465):589-593. doi: 10.1126/science.aay0934.
  PUBMED    31672889
REFERENCE   2  (bases 1 to 6374)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2019-11-1- 1"; volume: "366"; issue: 
            "6465"; pages: "589-593"
COMMENT     Sequence Label: pMtb CblA
FEATURES             Location/Qualifiers
     source          1..6374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..58
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        39..57
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    58..82
                     /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)."
     RBS             97..119
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             165..182
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1347..1365)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1361..1408
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1445..1900
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1532..1551)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1742..1763
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1992..2807)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1992..2807)
                     /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
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2715..2734
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2799..2818
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2929..3517
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3418..3437
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3671..3688
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3703..3842
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3768..3787)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3928..3950
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3944..4135)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4944..6026)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5988..6007
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6027..6104)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6310..6329
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
       61 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
      121 ataccatggg cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca
      181 gccatatgat ggccgcatcc cacgacgacg acaccgtcga cgggttggcg acggccgtgc
      241 gcggcggtga ccgtgcggcg ctgccacggg ccatcacact ggtcgagtcg acccgccccg
      301 accatcgtga gcaggcgcaa cagctgctgc tgcgattgct gccggactcc gggaacgccc
      361 atcgcgtcgg catcaccggg gtcccggggg tgggcaagtc gactgccatc gaggcgctgg
      421 gcatgcatct gatcgagcgc gggcatcggg tggcggtgct ggcggtcgac ccgtcgtcga
      481 cccgcacggg tggatcgatt cttggtgata aaacccggat ggcgcggctg gcggtgcacc
      541 cgaacgccta catccggccg tccccgacgt cgggaacgct gggtggggtg acgagggcca
      601 cccgggaaac ggtggtgctg ttggaggcgg ccggttttga tgtgatcctg atcgaaaccg
      661 tcggggtggg ccagtccgag gtcgcggtgg ccaacatggt cgacacgttc gtgttgctga
      721 ccttggcccg caccggtgat cagttgcagg gcatcaagaa gggcgtgctg gagctcgccg
      781 acatcgtggt ggtgaacaag gccgacgggg agcaccacaa agaggcccgg ctggccgccc
      841 gggagctgtc ggcggcgatc agattgatct atcctcgcga agcactgtgg cgcccaccgg
      901 tgctcaccat gagcgcggtg gagggcaggg gactggccga gctgtgggac accgtcgagc
      961 gtcatcgcca ggtgctcacc ggggccggcg aattcgacgc ccgtcggcgc gatcagcagg
     1021 tcgactggac ctggcagctg gttcgcgacg ccgtcctgga tcgggtgtgg tccaatccga
     1081 cggtgcgcaa ggtccgctcc gagctcgagc gtcgggtccg cgccggcgaa ctgaccccgg
     1141 ccctggcggc tcagcaaata ctggagatag ctaacctaac ggataggtaa gctagccaga
     1201 tatgcagcca tgaaggaggg gaatcaagag aaaatataca agaaaaatga cccatcggcc
     1261 gagtctgagg gactctgact accaccacca ccactgagat ccggctgcta acaaagcccg
     1321 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1381 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1441 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1501 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1561 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1621 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1681 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1741 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1801 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1861 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     1921 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1981 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2041 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2101 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2161 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2221 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2281 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2341 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2401 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2461 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2521 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2581 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2641 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2701 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2761 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     2821 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     2881 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2941 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3001 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3061 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3121 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3181 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3241 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3301 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3361 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3421 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3481 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3541 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3601 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3661 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3721 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     3781 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3841 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3901 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3961 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     4021 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4081 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4141 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     4201 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4261 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4321 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4381 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4441 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4501 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4561 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4621 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4681 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4741 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4801 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4861 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4921 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4981 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     5041 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     5101 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     5161 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     5221 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5281 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5341 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5401 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5461 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5521 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5581 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5641 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5701 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5761 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5821 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5881 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5941 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     6001 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     6061 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     6121 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     6181 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6241 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6301 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6361 gccggccacg atgc
//
