LOCUS       sequence-203921-        6639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Mycobacterium tuberculosis icl1 recombinant protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6639)
  AUTHORS   Bhusal RP, Patel K, Kwai BXC, Swartjes A, Bashiri G, Reynisson J, 
            Sperry J, Leung IKH
  TITLE     Development of NMR and thermal shift assays for the evaluation of 
            Mycobacterium tuberculosis isocitrate lyase inhibitors.
  JOURNAL   Medchemcomm. 2017 Oct 17;8(11):2155-2163. doi: 10.1039/c7md00456g. 
            eCollection 2017 Nov 1.
  PUBMED    30108733
REFERENCE   2  (bases 1 to 6639)
  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: 
            "10.1039/c7md00456g"; journalName: "Medchemcomm"; date: "2017-10-17-
            17"; volume: "8"; issue: "11"; pages: "2155-2163"
COMMENT     Sequence Label: pNIC28-Bsa4-ICL1
FEATURES             Location/Qualifiers
     source          1..6639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     44..63
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        44..62
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    63..87
                     /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             102..124
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             135..152
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             177..197
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1547..1564
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1617..1635)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1631..1678
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1715..2170
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1802..1821)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2012..2033
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2262..3077)
                     /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(2262..3077)
                     /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     2985..3004
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3069..3088
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3199..3787
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3688..3707
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3941..3958
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3973..4112
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4038..4057)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4198..4220
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4214..4405)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5214..6296)
                     /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     6258..6277
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6297..6374)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6580..6599
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
       61 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
      121 gagatataca tatgcaccat catcatcatc attcttctgg tgtagatctg ggtaccgaga
      181 acctgtactt ccaatccatg agtgtcgttg gaacgccaaa aagcgccgag caaattcagc
      241 aggaatggga cacaaaccca cgctggaaag acgtgacacg cacgtactcg gccgaagatg
      301 tggtggcgtt acagggctcg gttgtggaag aacacacgct ggcgcgccgt ggagccgaag
      361 tcctgtggga acaactgcat gacctggaat gggtgaatgc acttggcgcc cttactggta
      421 acatggctgt acagcaggtc cgcgcgggcc tgaaggcgat ttatctgagt ggatggcaag
      481 tggcaggcga tgctaacctg tcgggtcata cgtacccgga tcaaagctta tatccggcca
      541 attcagtccc acaagttgtt cgtcgtatca acaacgctct tcagcgcgct gatcagattg
      601 ccaagattga gggcgacaca agtgtcgaga attggctcgc cccgattgtg gcggacggag
      661 aagccggttt cggaggagca ctgaacgtgt atgaattaca gaaggcgctc attgccgcgg
      721 gcgtggcggg ctcacactgg gaagaccagc tggcgagcga aaagaagtgt ggccatctgg
      781 gcggtaaggt tttgattcct acccagcaac atatccggac tttaacctcc gcacgcctcg
      841 cagcggacgt ggccgatgtt ccgaccgtgg tgattgcacg cacagacgca gaggcggcga
      901 cattgattac atcagatgtt gacgagcgcg atcagccctt tatcactggg gaacgtacgc
      961 gcgagggctt ctaccgtact aagaatggca tcgaaccgtg catcgcccgt gccaaagcct
     1021 acgcgccctt cgcggatctg atttggatgg aaacgggcac gcctgacctc gaggccgctc
     1081 gtcaattttc ggaggctgtc aaagcagaat atccagatca gatgctggcg tataactgtt
     1141 ctccatcatt caattggaag aagcatctgg atgatgcgac aattgcgaaa ttccagaagg
     1201 agttggccgc gatgggcttc aagttccagt ttatcacctt agcgggcttc catgcgctta
     1261 attactccat gtttgatttg gcgtacggct atgcacagaa tcagatgagc gcttatgtag
     1321 aactgcagga gcgtgagttc gcggcggaag aacgtggcta tacggccacg aaacatcaac
     1381 gtgaggtagg agctggctat tttgaccgca tcgcgaccac tgtggatccc aacagttcaa
     1441 ccacagcgct taccggctcg actgaagaag gtcagtttca ctaacagtaa aggtggatac
     1501 ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca
     1561 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1621 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1681 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1741 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1801 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1861 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1921 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1981 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2041 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2101 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2161 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2221 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     2281 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2341 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2401 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2461 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2521 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2581 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2641 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2701 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2761 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2821 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2881 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2941 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     3001 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     3061 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     3121 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3181 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3241 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3301 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3361 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3421 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3481 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3541 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3601 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3661 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3721 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3781 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3841 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3901 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3961 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4021 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     4081 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     4141 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4201 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     4261 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4321 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4381 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4441 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4501 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4561 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4621 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4681 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4741 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4801 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4861 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4921 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4981 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     5041 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     5101 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     5161 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5221 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5281 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5341 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5401 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5461 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     5521 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5581 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5641 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5701 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5761 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5821 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5881 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5941 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     6001 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     6061 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     6121 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     6181 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     6241 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6301 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6361 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6421 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6481 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6541 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6601 gccagcaacc gcacctgtgg cgccggtgat gccggccac
//
