LOCUS       sequence-212890-        6837 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses M. tuberculosis Trigger Factor with a C-terminal His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6837)
  AUTHORS   Dotiwala F, Sen Santara S, Binker-Cosen AA, Li B, Chandrasekaran S, 
            Lieberman J
  TITLE     Granzyme B Disrupts Central Metabolism and Protein Synthesis in 
            Bacteria to Promote an Immune Cell Death Program.
  JOURNAL   Cell. 2017 Nov 16;171(5):1125-1137.e11. doi: 
            10.1016/j.cell.2017.10.004. Epub 2017 Oct 26.
  PUBMED    29107333
REFERENCE   2  (bases 1 to 6837)
  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: 
            "10.1016/j.cell.2017.10.004"; journalName: "Cell"; date: 
            "2017-11-16- 16"; volume: "171"; issue: "5"; pages: "1125-1137.e11"
COMMENT     Sequence Label: pET21a - Mtb-TF
FEATURES             Location/Qualifiers
     source          1..6837
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..93
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     177..196
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        177..195
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    196..220
                     /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             235..257
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             265..297
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1745..1762
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1815..1833)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1829..1876
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1913..2368
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2000..2019)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2210..2231
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2394..2498
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2499..2567,2568..3359)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2717..2736)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3530..4118
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4019..4038
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4272..4289
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4304..4443
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4369..4388)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4529..4551
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4545..4736)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5545..6627)
                     /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     6589..6608
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6628..6705)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
       61 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
      121 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
      181 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
      241 taactttaag aaggagatat acatatggct agcatgactg gtggacagca aatgggtcgc
      301 ggatccgaat tcgagctccg tcgacaagct tatgaagagc accgtcgagc agttgagccc
      361 cacccgggtt cgtatcaacg tggaggtgcc attcgccgag cttgagccgg atttccagcg
      421 ggcctacaaa gagctggcca aacaggtgcg gctgcccggc ttccggcccg ggaaggcgcc
      481 ggccaaacta ctcgaagccc gcatcggccg ggaggccatg ctggatcaaa tcgtcaacga
      541 tgcgctgccc agccggtacg gacaggcggt ggccgagtcg gatgtccaac cgctcggccg
      601 gcccaacatc gaggtgacca agaaggagta cggccaggac ctgcaattca ccgccgaggt
      661 cgacatccgc ccgaagatca gtctcccgga cctgagcgcg ctgacggtct cggtggatcc
      721 gatcgaaatc ggtgaggacg acgtcgacgc cgaactgcag tcgttacgta cccggttcgg
      781 caccctgacc gcggtggacc ggccggtggc cgtcggcgac gtcgtctcga tcgacttgtc
      841 tgccacggtc gacggagagg acataccgaa cgcagccgct gagggactct cccacgaggt
      901 cggctccggc cggctcatcg caggtctcga cgacgcggtt gttggtctgt ccgccgacga
      961 gtcccgggtc ttcaccgcca agctggcagc cggcgagcac gccgggcagg aagctcaggt
     1021 taccgtcacg gtcaggtcgg ttaaggagcg cgaactacca gagcccgacg acgaattcgc
     1081 gcagttagcc agcgagttcg acagcatcga cgaattgcgg gccagcctca gcgaccaggt
     1141 gcgccaggcc aagcgcgccc agcaggccga gcagattcga aacgccacca tcgatgcgct
     1201 actcgaacag gtcgacgtgc cgttgccgga gtcgtatgtg caggcccaat tcgacagcgt
     1261 gctgcacagc gcgctcagcg gtcttaatca cgacgaagcc cggttcaatg agttgctcgt
     1321 cgagcaaggc tcgtcacgcg cggcgttcga tgccgaggcg cgcaccgcct cagaaaagga
     1381 cgtcaagagg cagctgttgc tagacgccct ggccgatgag ctgcaggtcc aagttggcca
     1441 ggatgatctg accgaacgac tggtgacgac gtctcggcaa tacggcatcg agccgcagca
     1501 gctgttcggc tacctccaag agcgcaacca gctgccgacc atgttcgctg acgtgcggcg
     1561 cgagctggcg atcagggccg cagtggaggc ggcgacggtc accgacagtg acggaaacac
     1621 gatcgatacc agtgagttct tcggcaagcg tgtgtcggcc ggtgaggctg aggaggccga
     1681 accggcagac gagggtgccg cgcgggcggc gtccgacgaa gcgacaacgg cggccgcact
     1741 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     1801 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1861 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     1921 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1981 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2041 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2101 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2161 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2221 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2281 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2341 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     2401 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     2461 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     2521 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     2581 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     2641 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     2701 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     2761 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     2821 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     2881 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     2941 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3001 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     3061 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     3121 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     3181 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     3241 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     3301 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     3361 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     3421 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     3481 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3541 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3601 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3661 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3721 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3781 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3841 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3901 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3961 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4021 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4081 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4141 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4201 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4261 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     4321 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     4381 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     4441 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4501 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4561 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     4621 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     4681 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     4741 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     4801 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     4861 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     4921 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     4981 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     5041 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     5101 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     5161 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     5221 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     5281 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     5341 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     5401 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     5461 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     5521 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5581 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     5641 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     5701 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     5761 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     5821 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     5881 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     5941 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     6001 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     6061 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     6121 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     6181 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     6241 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     6301 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     6361 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     6421 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     6481 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     6541 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     6601 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     6661 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     6721 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     6781 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggcca
//
