LOCUS       sequence-176695-        6755 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses recombinant 6HIS tagged T. cruzi thiol dependent reductase
            in E. coli upon IPTG induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6755)
  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 6755)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 - TDR
FEATURES             Location/Qualifiers
     source          1..6755
                     /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             1399..1416
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1469..1487)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1483..1530
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1567..2022
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1654..1673)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1864..1885
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2048..2152
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2153..2221,2222..3013)
                     /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(2371..2390)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3184..3772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3673..3692
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3926..3943
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3958..4097
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4023..4042)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4183..4205
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4199..4390)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5199..6281)
                     /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     6243..6262
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6282..6359)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6565..6584
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6668..6687
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6668..6686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6687..6711
                     /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             6726..6748
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcat gaaggctttg
       61 aaacttttta aagaccgatt atgccccttt tgccaacgcg tgttgatcac ggcgaaggaa
      121 aagcgtgtca cgttggagga ggtggaggtt cctcttggcg atgacatgcc gcagtggtac
      181 aaggagctga acccacggga gacggttcca acgcttcagg tggatggaaa aaaatgcatg
      241 atcgagtcag atctcatttc acggtacatt gaccgcattt cttctcccgc gaacgccctt
      301 atggggtcaa gcccctacca acggcaccgc gtggagtttt ttctgggtga gatcggcgat
      361 cttgtcaaag catattttgg ccttgttagg gaccctttca atgaggagaa gcggaagtcc
      421 gtggataaca acaccgcata tatcgaggat atcattgcag aacatcaggg ggacggacca
      481 tattttcttg atgacacgtt ttccatggct gaagttatgg tcgttccatt tcttgcttgt
      541 ttccgtccag ttttaagtta ctattgtggt tatgacatct tccataatgc tccccgtctc
      601 aagaaaatgt acgtgacgtc catgcagcgt acaactgtga aggaaacgat ttcaaagcca
      661 gaagaataca ttattggctt taagagtaag gtgccaaaat cgcacgtcac atggtcattg
      721 gctcctggct atgtactttt tgttaacaag tattctccat tttcggatcg ccctcgtttg
      781 gcatgtgcac tgaagaatat tgatttgccc atgcttgaaa ttgatttgaa acagttgccc
      841 ccgtggttcc gatggtttaa ccagcgtgaa actgtgccaa ctttgcttac cccacagggt
      901 acctacgttc atgagtccca acttattgtt cattatcttg atgatgggtt cccagagcat
      961 gggcctgcgc tacttcccaa ggatgccgat ggatcgtacc atgtgaggtt tgttgaatct
     1021 aatgtggatt actttatgga tgccatgtat tctttcataa aggatccaaa aaacatgaat
     1081 gcaaaggaag aatttgattg ggcagcaggt gagcttgaga aattattggc ggaacatcag
     1141 tttggggagg gtccattttt tggtggagcc acaatgaacg ctgctgacgt ttctcttgtg
     1201 cccatgctgg tgcacctgaa ggcctgcact ccagaactga cggaaggcca ggatttgctt
     1261 gcaaattaca agctgttggc cgccgccgcg gaagcgggtt tgacgagtga ggcaggcaaa
     1321 aaggtctttt tgtcactctc ggaatactcg agtatataca agacgttttt gcgtccatcg
     1381 tctgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     1441 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1501 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1561 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1621 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1681 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1741 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1801 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1861 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1921 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1981 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     2041 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2101 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2161 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2221 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2281 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2341 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     2401 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2461 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     2521 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     2581 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     2641 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     2701 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     2761 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     2821 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     2881 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2941 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3001 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3061 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3121 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3181 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3241 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3301 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3361 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3421 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3481 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3541 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3601 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3661 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3721 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3781 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3841 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3901 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3961 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     4021 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     4081 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4141 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4201 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     4261 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     4321 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     4381 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     4441 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     4501 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4561 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4621 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4681 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4741 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4801 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     4861 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     4921 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     4981 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     5041 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     5101 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     5161 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5221 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5281 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     5341 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     5401 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     5461 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     5521 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5581 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5641 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5701 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5761 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5821 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5881 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5941 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     6001 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     6061 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     6121 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     6181 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     6241 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     6301 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     6361 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     6421 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     6481 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     6541 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     6601 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     6661 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     6721 ataattttgt ttaactttaa gaaggagata tacat
//
