LOCUS       sequence-176687-        6593 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses recombinant 6HIS tagged T. cruzi NAD-FAD–dependent 
            dehydrogenase in E. coli upon IPTG induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6593)
  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 6593)
  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 - NFDD
FEATURES             Location/Qualifiers
     source          1..6593
                     /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             1237..1254
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1307..1325)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1321..1368
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1405..1860
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1492..1511)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1702..1723
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1886..1990
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1991..2059,2060..2851)
                     /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(2209..2228)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3022..3610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3511..3530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3764..3781
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3796..3935
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3861..3880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4021..4043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4037..4228)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5037..6119)
                     /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     6081..6100
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6120..6197)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6403..6422
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6506..6525
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6506..6524
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6525..6549
                     /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             6564..6586
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgcgcca ggaagaagaa
       61 agcaaacaga agaccctaga cgccacggtg cgtgatgaca atccgtatag ccggctcatg
      121 gcactgcagc gaatgggtgt tgtagacaac tatgaggcca tccgccaaaa gtctgtggcg
      181 attataggcg ccggtggggt cggaagtgtt gtggcagaga tgttgacacg ttgtggtatt
      241 tcaaagattc taatatttga ctacgacaag gtggagcttg cgaacatgaa tcgtcttttt
      301 taccgacctg agcagaaggg catgacgaag gtgcttgcgg caaaacagac tttggaggat
      361 atcaaccccg acacggagat tgtgccatac gccttcagta ttacttccac ggaacattgg
      421 caggattttg tggatgccct gacgaagaat ggtggagtac agccgaattc acctgtggat
      481 ctgttactct gttgtgtaga caactttcag gcccgcctta ctgtgaatta cgcctgttta
      541 cttcataaca ttccatggat ggaaagtggc gttgcagaga atgccgtcag tggtcatatc
      601 cagctgcttc ttccaggcgt gacgccttgc tacgagtgct gccccccgct ggtggtggcg
      661 acaggaatgc cggaggcaaa acgtgagggt gtctgtgcgg cgtctcttcc caccaccatg
      721 ggcatcgtgg cgggatttat ggcgcagaat acactgaaat atctactgaa ttttggaact
      781 gtgagcgagt acgttggata tgacgcaatg cgggatcatt tcccctctat cacgattaaa
      841 gcaaacccgg agtgccgcaa tgagacatgt gtgcagcgac aacaagagta cgctgcgcga
      901 cgggccgcga tgggcgatgc ggcccaccca ctacatcagg cgaataaaca acgtgaggag
      961 cgagaggcaa aggaacgagc tgcagcgagg gcgaaggcga cagcgtcggc agccgaatgg
     1021 ggtattgtaa ttgaggcccg cggcaaggag gacttggcgg tgcatgcgcc acagcctgag
     1081 ggcgtggaag ccgtcagtgt ggagtacgcg tacagcagcg gtgcggggaa tgcggcggag
     1141 cacgcacagg aggaattcgt gatgacggac ggtggagagt cactcgaggc attgatggct
     1201 aaaatgaagg ctctgcaaca cgcggccgca ctcgagcacc accaccacca ccactgagat
     1261 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1321 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1381 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1441 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1501 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1561 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1621 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1681 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1741 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1801 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1861 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1921 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1981 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2041 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2101 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2161 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2221 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2281 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2341 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2401 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2461 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2521 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2581 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     2641 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     2701 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     2761 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     2821 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2881 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     2941 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3001 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3061 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3121 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3181 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3241 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3301 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3361 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3421 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3481 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3541 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3601 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3661 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3721 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3781 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3841 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3901 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3961 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4021 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4081 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4141 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4201 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     4261 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4321 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4381 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4441 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4501 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4561 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4621 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4681 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4741 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4801 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4861 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4921 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4981 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     5041 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5101 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5161 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5221 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5281 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     5341 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5401 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5461 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5521 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5581 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5641 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5701 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5761 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5821 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5881 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5941 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6001 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     6061 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     6121 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     6181 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     6241 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     6301 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     6361 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     6421 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     6481 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     6541 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata cat
//
