LOCUS       V010364                 8673 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V010364
VERSION     V010364
KEYWORDS    pRSIT16-U6Tet-sh-HTS6-CMV-TetRep-2A-TagRFP-2A-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8673)
  AUTHORS   Cellecta
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8673)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label="RSV promoter"
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label="5' LTR (truncated)"
                     /note="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1078..1311
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             1496..1540
                     /label="gp41 peptide"
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
                     al., 2013)"
     CDS             1689..1730
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     protein_bind    2020..2037
                     /gene="tetO"
                     /label="tetracycline repressor TetR binding site"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2046..2064
                     /label="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    2066..2095
                     /label="cloning site"
                     /note="cloning site"
                     /note="shRNA cloning site"
     misc_feature    2123..2238
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1 (lacking the first T)"
     promoter        2387..2590
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             2664..3284
                     /label="TetR"
                     /note="tetracycline repressor TetR"
     CDS             3291..3344
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid
                     protein"
                     /label="2A peptide from Thosea asigna virus capsid protein"
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3351..4061
                     /label="TagRFP"
                     /note="monomeric derivative of red fluorescent protein from
                     Entacmaea quadricolor (Merzlyak et al., 2007)"
     CDS             4080..4133
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid
                     protein"
                     /label="2A peptide from Thosea asigna virus capsid protein"
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4140..4736
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     misc_feature    4752..5340
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             5414..5647
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5719..5853
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      5880..6015
                     /label="SV40 ori"
                     /note="SV40 origin of replication"
     primer_bind     complement(6048..6064)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(6072..6088)
                     /label="lac operator"
                     /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)."
     promoter        complement(6096..6126)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6141..6162)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6450..7038)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(7212..8069)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(8070..8174)
                     /label="AmpR promoter"
     primer_bind     8648..8664
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccact gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     1801 atggccgccc ccttcaccga gggcctattt cccatgattc cttcatattt gcatatacga
     1861 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
     1921 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     1981 tttaaaatgg actatcatat gcttaccgta acttgaaagc cctatcagtg atagagattt
     2041 atatatccct atcagtgata gagacaccgg agtcttcttt tttgaagaca cttcggactg
     2101 tagaactctg aacctcgagc aatttaaaag aaaagggggg attggggggt acagtgcagg
     2161 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     2221 tacaaaaatt caaaatttct gcgttgttgt cggtgctcgt tctctgctct tcacgctact
     2281 gaattcatca ccggttcttc gaatctagta ttatgcccag tacatgacct tatgggactt
     2341 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     2401 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     2461 cattgacgcc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     2521 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtgga aggtttatat
     2581 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
     2641 cctccataga agatctagac accatgtcca gactggacaa gagcaaagtc atcaactctg
     2701 ccttggagct cctgaatgaa gttggcattg agggcttgac caccaggaag ctggcccaga
     2761 agctgggtgt ggagcagcct accctgtact ggcatgtgaa gaacaagagg gctctgcttg
     2821 atgccctggc cattgagatg ttggacaggc accacaccca cttctgccct ctggaagggg
     2881 agtcctggca ggacttcctg aggaacaatg ccaagagctt cagatgtgcc ttgctctccc
     2941 accgggacgg tgccaaagtt cacttgggca ccaggcctac agagaagcag tatgagaccc
     3001 tggagaacca gctggcattc ctgtgccaac aaggcttctc cctggaaaat gccttgtatg
     3061 ccctctctgc tgtgggccac ttcaccttgg gctgtgtgct ggaggaccag gagcaccaag
     3121 ttgccaagga ggagagggag acccccacca ctgactccat gccaccactg ctgcggcaag
     3181 ctattgagtt gtttgaccac caaggggctg agcctgcatt cctgtttggc ctggaactga
     3241 tcatctgtgg cctggaaaag cagctgaaat gtgagtctgg ctctttcgaa gagggcagag
     3301 gaagccttct aacatgcggt gacgtggagg agaatcccgg cccttccgga atggtgtcta
     3361 agggcgaaga gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtga
     3421 acaaccacca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga
     3481 ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta
     3541 ccagcttcat gtacggcagc agaaccttca tcaaccacac ccagggcatc cccgacttct
     3601 ttaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac gaggacgggg
     3661 gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca
     3721 agatcagagg ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa acactcggct
     3781 gggaggccaa caccgagatg ctgtaccccg ctgacggcgg cctggaaggc agaagcgaca
     3841 tggccctgaa gctcgtgggc gggggccacc tgatctgcaa cttcaagacc acatacagat
     3901 ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac cacagactgg
     3961 aaagaatcaa ggaggccgac aaagagacct acgtcgagca gcacgaggtg gctgtggcca
     4021 gatactgcga cctccctagc aaactggggc acaaacttaa tgctagcacc ggtagatccg
     4081 agggcagagg aagccttcta acatgcggtg acgtggagga gaatcccggc ccttccggga
     4141 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     4201 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     4261 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     4321 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     4381 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     4441 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     4501 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca
     4561 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     4621 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     4681 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa
     4741 tctaggtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     4801 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     4861 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     4921 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     4981 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     5041 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     5101 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     5161 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     5221 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     5281 ttccgcgtct ccgccttcgc cctcagacga gtcggatctc tctttgggcc gcctccccgc
     5341 ctggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     5401 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gataagatct gctttttgct
     5461 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     5521 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     5581 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     5641 tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga
     5701 atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     5761 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     5821 aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca
     5881 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     5941 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     6001 gcttttttgg aggcctagac ttttgcagag accaaattcg taatcatgtc atagctgttt
     6061 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     6121 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     6181 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6241 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     6301 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6361 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6421 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6481 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6541 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6601 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6661 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6721 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6781 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6841 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6901 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6961 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     7021 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     7081 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     7141 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     7201 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     7261 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     7321 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     7381 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7441 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7501 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7561 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7621 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7681 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7741 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7801 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7861 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7921 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7981 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     8041 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     8101 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     8161 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     8221 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     8281 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     8341 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     8401 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     8461 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc
     8521 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     8581 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     8641 cgacgttgta aaacgacggc cagtgccaag ctg
//
