LOCUS       sequence-176690-        6736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  [Edit] Expresses recombinant 6HIS tagged T. cruzi trypanothione 
            reductase protein in E. coli upon IPTG induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6736)
  AUTHORS   Dotiwala F, Mulik S, Polidoro RB, Ansara JA, Burleigh BA, Walch M, 
            Gazzinelli RT, Lieberman J
  TITLE     Killer lymphocytes use granulysin, perforin and granzymes to kill 
            intracellular parasites.
  JOURNAL   Nat Med. 2016 Feb;22(2):210-6. doi: 10.1038/nm.4023. Epub 2016 Jan 
            11.
  PUBMED    26752517
REFERENCE   2  (bases 1 to 6736)
  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: "10.1038/nm.4023"; 
            journalName: "Nat Med"; date: "2016-02"; volume: "22"; issue: "2"; 
            pages: "210-6"
COMMENT     Sequence Label: pET21a - TR
FEATURES             Location/Qualifiers
     source          1..6736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..33
                     /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             1380..1397
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1450..1468)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1464..1511
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1548..2003
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1635..1654)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1845..1866
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2029..2133
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2134..2202,2203..2994)
                     /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(2352..2371)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3165..3753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3654..3673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3907..3924
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3939..4078
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4004..4023)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4164..4186
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4180..4371)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5180..6262)
                     /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     6224..6243
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6263..6340)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6546..6565
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6649..6668
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6649..6667
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6668..6692
                     /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             6707..6729
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgaagcc gtgaattcaa
       61 gacgtcggac gctgaaatct aaatgccctt tggcggccat ttttctttcg ataccgcttt
      121 tttttatggt attactttta acgagcattg tttttgcgga ggcacttccg tttacgaaat
      181 tctctggcgt tgtggaacta acgcctgcaa catttaagaa ttttgtgtct tcccacaagc
      241 cagtgtacat tcttttttat gccccgtggt gcggtcactg ccgcagaatt cacccagagt
      301 gggagaaatt tgctcagtct gcctacggca ccgtccgcgt gggtgccatc aacgccgacg
      361 aacactcgca gattgcaggg cagtttggca ttcgtggttt tccaaccata aagtactgga
      421 acgtgggtga aaaggacatc aacaagccac aggagtacaa tggaccgcga caggcgaagt
      481 cacttcaagc caacgcgatg aatcaaatca cttcctccgg gataaaagca attacgagca
      541 gtgatgcctt gcgggaggca gtacaaaagg cacctgagaa gaaaattgtg gttctcttct
      601 cttccaagcc acgtattccc gccatttttg ctgttttgtc acattcccca cgtttgaaat
      661 ctatgccgtt ttattttgtt ggtgaaaatg ctaaaaaaga agtctcggag gaatttggag
      721 tgcaggaacg cccgagcatc gctgttctca acgccacgga aagcgacatc aagactgtat
      781 tatacccggg aaaacagatt gcctatgaac ccattgcgaa gttcttgttg gcatgcgcaa
      841 cagatgatgc cgataatttg gatgccgcga aaggaaatgc aggggagcat ccacaagaaa
      901 acgacagtgg caagggcaag aataccgctg cggctggtgg tgggacaccc acgccagcat
      961 tacccgtgcg ccccgtacga ctaaccgcac agttgttggc ggacttctgc ttaccggcga
     1021 cactgcgcgt acatgaacgg gcaccgctgt gtgtgttgtc gtttacgtcc gcgtttactc
     1081 ttgagcgtgt tcacgagatg tttcgcaatg aacccctttt attcttcgag gcagatgccg
     1141 agcgtgatgt cattctttct gcccttcgga atgatcttgg tctggaagag gtggccaaca
     1201 gtgttgaggg tggtgccgct catcagatgt tattgttacg ggcagcgaag cgtgacacga
     1261 tccgctacag gcttcttgac gggatagaga cagtggaagg tgcaaatgaa gtactgcaga
     1321 gggtgctgtc gggagatttg cagctcggga aaaaagtgct gcaggcggcc gcactcgagc
     1381 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     1441 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     1501 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     1561 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1621 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1681 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1741 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1801 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1861 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1921 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1981 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     2041 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2101 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2161 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2221 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2281 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2341 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2401 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2461 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2521 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2581 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2641 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     2701 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2761 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2821 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2881 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2941 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3001 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3061 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3121 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3181 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3241 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3301 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3361 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3421 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3481 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3541 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3601 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3661 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3721 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3781 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3841 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3901 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3961 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     4021 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4081 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4141 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4201 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4261 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4321 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4381 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4441 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4501 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4561 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4621 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4681 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4741 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4801 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     4861 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4921 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     4981 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     5041 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     5101 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5161 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5221 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5281 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5341 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5401 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5461 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5521 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5581 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5641 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5701 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5761 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5821 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5881 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     5941 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     6001 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     6061 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     6121 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     6181 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6241 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     6301 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6361 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6421 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6481 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6541 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6601 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     6661 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     6721 agaaggagat atacat
//
