LOCUS       sequence-176686-        6356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses recombinant 6HIS tagged T. cruzi Glutathione reductase 
            protein in E. coli upon IPTG induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6356)
  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 6356)
  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 - GR
FEATURES             Location/Qualifiers
     source          1..6356
                     /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             1000..1017
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1070..1088)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1084..1131
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1168..1623
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1255..1274)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1465..1486
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1649..1753
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1754..1822,1823..2614)
                     /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(1972..1991)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2785..3373
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3274..3293
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3527..3544
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3559..3698
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3624..3643)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3784..3806
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3800..3991)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4800..5882)
                     /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     5844..5863
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5883..5960)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6166..6185
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6269..6288
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6269..6287
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6288..6312
                     /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             6327..6349
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcat ggttttgggt
       61 aagggatcaa ttggccgaaa gttgctgggg gcgacaggtc tgatcggctc actggcgatt
      121 ggcggcgcct attttacgta tcagcgacga ctgaaggaaa accgcagctg cacggctgaa
      181 cagttcaacg cggcacaaaa ccaggaagaa ctgtcttggg ctctttcaca cttgaggaat
      241 ccgaaggagc acccgcagat tcttctctat cgatattcca cttgcccctt ttgtgggacg
      301 gcgaaggccc tgctggacta cgccaaaatt ccacacgaat gtgttgaggt ggagccaatg
      361 tttaaaaagg agatttcctc atttgcgtac aaaaaagtac cacagttgca gtttaatgta
      421 cacggataca atggcccact tgtcgtggac tctgaaatta tcgtcagtac tcttgcaaag
      481 catgtgggta tggagaagca gctgaaggat ccggaagttg tgaagtggcg tgagtgggcc
      541 cgcggaccaa tggtacgcct tttgactctg gaatttaact cttctcttta cagggcgtgg
      601 tgcggttatt cctacataaa taacattgac acgatcccgt attcgaacaa gttgttccta
      661 aagatagtgg gtgcacctgt gatgtacctc gtatctcaat acatcactag gccccgactc
      721 cttaaaagcg gtcatctaca cgagggggaa gatgtaaaag gaagacttca tggtgagata
      781 aacaccttta ttgaaaaagc ccttttgggt gggaagaaaa agtttcacgg tggctcaaaa
      841 ccagatcttg cggatttgga cacttacggt gttcttcaat ctgttagagg acaccgcgtc
      901 tatgaggaaa ttgttcaatc tacgccaatt aaaccatggt tggatagtat ggacaaggag
      961 gttggccacg tgagccatga tggggcggcc gcactcgagc accaccacca ccaccactga
     1021 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1081 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1141 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1201 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1261 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1321 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1381 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1441 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1501 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1561 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     1621 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1681 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1741 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1801 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     1861 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     1921 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     1981 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2041 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2101 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2161 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2221 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2281 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2341 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2401 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2461 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2521 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2581 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2641 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2701 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2761 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2821 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2881 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     2941 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3001 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3061 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3121 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3181 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3241 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3301 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3361 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3421 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3481 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3541 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3601 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3661 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     3721 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3781 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     3841 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     3901 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     3961 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     4021 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     4081 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     4141 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     4201 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     4261 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4321 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4381 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4441 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     4501 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     4561 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     4621 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     4681 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4741 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4801 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4861 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     4921 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     4981 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     5041 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     5101 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5161 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5221 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5281 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5341 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5401 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5461 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5521 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5581 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5641 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5701 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     5761 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     5821 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     5881 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     5941 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     6001 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     6061 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6121 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     6181 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6241 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     6301 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacat
//
