LOCUS       sequence-93746-e        6051 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For use in trypanosome cell lines already expressing T7 polymerase 
            and Tet-repressor. Tetracycline induction results in the production 
            of dsRNA targeting VSG221..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6051)
  AUTHORS   Sheader K, Vaughan S, Minchin J, Hughes K, Gull K, Rudenko G
  TITLE     Variant surface glycoprotein RNA interference triggers a 
            precytokinesis cell cycle arrest in African trypanosomes.
  JOURNAL   Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8716-21. Epub 2005 Jun
            3.
  PUBMED    15937117
REFERENCE   2  (bases 1 to 6051)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2005-06-14"; volume: "102(24)"; pages: 
            "8716-21. Epub 2005 Jun 3"
COMMENT     Sequence Label: MC177VSG221RNAi
FEATURES             Location/Qualifiers
     source          1..6051
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(595..642)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      complement(728..775)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        967..985
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    992..1010
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1868..1886
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(1893..1911)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2163..2210
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2296..2343
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             2812..3186
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     primer_bind     complement(3596..3615)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3826..3845)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3945..3967
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4005..4023)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4091..4195
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4196..4264,4265..5056)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4414..4433)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5227..5815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5716..5735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5969..5986
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gaattcgagc tctttaatgg tccttatacg aacgaatatt aaacaatgcg cagttaacgc
       61 tattatacac aataactttt aatgtctgcc atattaatta caagtgtgca acattaaata
      121 caagtgtgta acgttaattt gcaagtttga aacaatgttc tttagtgttt aatgtgtgca
      181 acaaagctat ttaatggtcc ttatacgaac gaatattaaa caatgcgcag ttaacgctat
      241 tatacacaat aacttttaat gtgtgccata ttaattacgg atcgcggccg cgatccgtgt
      301 gcaacattaa atacaagtgt gtaacattaa tttgcaagtt tgcaacaatg ttctttagtg
      361 tttaatgggt gcaacaaagc tatttaatgg tccttatacg aacgaatatt aaacaatccg
      421 cagttaacgc tattatacac aataactttt aatgtgtccc atattaatta caagtgtgca
      481 acattaaata caagtgtgta acgttaattt gcaagtttgc aacaatgttc tttagtgttt
      541 aatgggtacc gaattcctgc aggggcccta tccggatata gttcctcctt tcagcaaaaa
      601 acccctcaag acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc
      661 agcagccaac tcagcttcct ttcgggcttt gttagcagcc ctatccggat atagttcctc
      721 ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt
      781 gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca gcctgcagcc
      841 caggaccgaa tactaataac gacacttgcg gtcaaaaagt agaagaacaa atgctcaacg
      901 atgagtgaat caggttaggg tagttggaaa attatacaga atgtctttgg caacacaccg
      961 cctgattaat acgactcact atagggagat ctccctatca gtgatagaga tctagccgcg
     1021 gtggcggccg gccgctctag aagctttttg gcaacctctt tgccaggtct ccgaggagct
     1081 agacgaccaa ccgaagggtg cgttgtttac gctgcaagca gcggcgagca aaatccagaa
     1141 aatgagggac gcggcactgc gagcaagtat atacgctgaa ataaatcacg gcaccaacag
     1201 ggccaaggca gccgttatag tcgccaacca ctatgccatg aaagctgata gcggcctaga
     1261 ggccctaaaa caaacgttaa gcagccaaga ggtaacagct actgcaacag cgagctacct
     1321 aaaaggaaga atagacgaat acttaaatct ccttctacaa acaaaggaga gcggcaccag
     1381 cggctgcatg atggacacca gcggaacaaa cacggtaacg aaggccggcg gcaccatcgg
     1441 aggcgttcct tgcaagctgc agttgtcgcc gatacagccg aagcgacccg cagcgaccta
     1501 cctaggtaaa gcgggctacg taggcctaac acgacaagca gatgcagcca acaatttcca
     1561 cgataacgac gccgaatgca ggctagccag tgggcacaac accaacggcc tcggcaaaag
     1621 cggccagctt tctgcagcgg tcactatggc ggccggctat gtcacagtag cgaacagcca
     1681 aacagccgtc acggtccagg cgctcgatgc attacaggaa gcgagcggag cagcgcacca
     1741 accgtggatc gacgcctgga aggccaagaa agcgctaaca ggagcagaaa ccgctgagtt
     1801 cagaaacgaa acagccggaa tagctggcaa aacaggcgtt accaactcga gggggggccc
     1861 ggggagatct ctatcactga tagggagatc tccctatagt gagtcgtatt aatcaggtaa
     1921 cggtgtgttg ccaaagacat tctgtataat tttccaacta ccctaacctg attcactcat
     1981 cgttgagcat ttgttcttct actttttgac cgcaagtgtc gttattagta ttcggtcctc
     2041 cgcatgattg aaatagtgcc accagttgtg tttgatgcgt ttgttatcta tgcagtattc
     2101 tgcaggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     2161 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2221 gaactatatc cggatagggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     2281 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     2341 ttgctgaaag gaggaactat atccggattg ggcccctgca ggcatgcaag ctagctttcc
     2401 acccagcgcg ggtgcattct ggctcttata tatacttatt gtcatgacag agtatattgt
     2461 actgtgttga taagggacgg gtaactgtat tgaagagccg atgcttttga catgttagat
     2521 ataatatgtt ttattgtaaa gtcaatacaa cacacaatag gataataatg ataaagttaa
     2581 aaaagtatat atagtaatag aaatatatct tatataggaa agattaagca gtaaaagtag
     2641 cgcttacggc gtacggagca ggagagcaac tgaccgctct cagacccggg cacagcaagg
     2701 tcttctgaaa ttcatgtttt tttttttttt actctgcatt gcagtctccg ctcttattta
     2761 gttttgcttt acgtaaggtc tcgttgctgc cataaaataa gctatatcac catggccaag
     2821 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     2881 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     2941 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     3001 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     3061 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     3121 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     3181 gactgaccga cgccgaccaa caccgccggt ccgacggcgg cccacgggtc ccaggcctcg
     3241 gagatcctaa caccgggttg tgtggccaaa attgttctgt agtcgctgtg agttgacacg
     3301 gctagtgctt atgattttcc tcgcgtgtgg tgcctgtact cagccctatg ccttatttgc
     3361 aacacattta cgtacagcgc acaagagaag agaagatcac ttgaagataa taaatatagg
     3421 gttgtaggca tcttgtttaa ctcaaatttt ctcgtcttgg tgtgtcgaca tgattgaaat
     3481 agtgccacca gttgtgtttg atgcgtttgt tatctatgca gtattcctag ccagatcctc
     3541 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
     3601 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
     3661 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
     3721 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     3781 tgcaggagtc gcataaggga gagcgtcgat atggtgcact ctcagtacaa tctgctctga
     3841 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3901 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3961 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     4021 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     4081 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4141 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4201 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4261 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4321 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4381 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4441 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4501 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4561 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4621 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4681 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4741 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4801 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4861 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4921 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4981 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5041 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5101 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5161 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5221 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5281 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5341 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5401 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5461 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5521 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5581 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5641 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5701 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5761 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5821 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5881 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5941 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     6001 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca g
//
