LOCUS       sequence-176704-        6212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses recombinant 6HIS tagged T. cruzi NADP-dependent 
            oxidoreductase in E. coli upon IPTG induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6212)
  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 6212)
  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 - NDH
FEATURES             Location/Qualifiers
     source          1..6212
                     /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             856..873
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(926..944)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      940..987
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1024..1479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1111..1130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1321..1342
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1505..1609
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1610..1678,1679..2470)
                     /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(1828..1847)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2641..3229
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3130..3149
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3383..3400
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3415..3554
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3480..3499)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3640..3662
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3656..3847)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4656..5738)
                     /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     5700..5719
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5739..5816)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6022..6041
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6125..6144
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6125..6143
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6144..6168
                     /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             6183..6205
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgtcagg acaaaaaaga
       61 gttttgtcag ggagggtggc agttgtcacg gggtcaagaa agggtatcgg tctcggcatt
      121 gcgatgcggc tggcgatggc tggagcggat gttgttctta atggcaggcg ccagagtcca
      181 gaggattcgg cgattgtcga aaaagtggcc gcctacggca cgcgcgttcg ctgctttgct
      241 gccgacttga aagatcgtgc acaggtcgaa gcgctgataa agtttacgga gaaggagctg
      301 ggcgccgtgg aaattctggt caataacgcc ggtattcagc acgtctcccc agtggaaaca
      361 tttccttccg acaagtggga cgaaatcatc gctctgaatc tcacatccgc ttttcatgcg
      421 acgcagctgt gtctgccggg catgcgtcag cgcgggtggg gccgcattat taacattgcc
      481 tcggttcaag ggttggtggg ttcggtgaat aaatctgctt actgtgctgc caagcacggt
      541 ctcattggat tcaccaaagt tgtggccctg gaaactgcca cgacaggaat tacttgcaac
      601 gcgatatgtc ccgggtacgt ctatacaccc ttggtggagg aacaaatcaa agcggttgct
      661 gaagcaaaat acggcggtga catggaggcc gcaactcagg catttctctg tgaaaaacaa
      721 ccagcaaaag catttgtgac tgtcgagcag gttggagacg ctgcggtgtt tctcgcaagt
      781 ccaggggcag acctgattcg cggcaccaca atcaccgtgg atggtggatg ggtcgcccaa
      841 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
      901 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      961 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1021 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1081 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1141 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1201 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1261 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1321 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1381 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1441 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1501 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1561 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1621 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1681 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1741 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1801 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     1861 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1921 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1981 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2041 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2101 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2161 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2221 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2281 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2341 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2401 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2461 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2521 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2581 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2641 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2701 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2761 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2821 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2881 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2941 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3001 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3061 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3121 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3181 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3241 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3301 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3361 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3421 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3481 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3541 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3601 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3661 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3721 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3781 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3841 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3901 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3961 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4021 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4081 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4141 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4201 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4261 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4321 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4381 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4441 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4501 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4561 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4621 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4681 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4741 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4801 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4861 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4921 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4981 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5041 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5101 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5161 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5221 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5281 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5341 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5401 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5461 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5521 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5581 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5641 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5701 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5761 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5821 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5881 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5941 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     6001 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     6061 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     6121 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     6181 attttgttta actttaagaa ggagatatac at
//
