LOCUS       V010365                 8706 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V010365
VERSION     V010365
KEYWORDS    pRSIT12-U6Tet-sh-HTS4-CMV-TetRep-2A-TagRFP-2A-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8706)
  AUTHORS   Cellecta
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8706)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8706
                     /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    2139..2256
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        2420..2623
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             2697..3317
                     /label="TetR"
                     /note="tetracycline repressor TetR"
     CDS             3324..3377
                     /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             3384..4094
                     /label="TagRFP"
                     /note="monomeric derivative of red fluorescent protein from
                     Entacmaea quadricolor (Merzlyak et al., 2007)"
     CDS             4113..4166
                     /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             4173..4769
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     misc_feature    4785..5373
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             5447..5680
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5752..5886
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      5913..6048
                     /label="SV40 ori"
                     /note="SV40 origin of replication"
     primer_bind     complement(6081..6097)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(6105..6121)
                     /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(6129..6159)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6174..6195)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6483..7071)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(7245..8102)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(8103..8207)
                     /label="AmpR promoter"
     primer_bind     8681..8697
                     /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 aacctctgaa ttctcactcg agccacaatt ttaaaagaaa aggggggatt
     2161 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     2221 gaattacaaa aacaaattac aaaaattcaa aattttgcgg ttgtgctttt tgcgtgtaaa
     2281 tctcggtggt cgccgtatca tggcttgtgc gatgcgttgg ctaccggttc ttcgaatcta
     2341 gtattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     2401 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     2461 gtttgactca cggggatttc caagtctcca ccccattgac gccaatggga gtttgttttg
     2521 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     2581 gggcggtagg cgtgtacggt ggaaggttta tataagcaga gctcgtttag tgaaccgtca
     2641 gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagatcta gacaccatgt
     2701 ccagactgga caagagcaaa gtcatcaact ctgccttgga gctcctgaat gaagttggca
     2761 ttgagggctt gaccaccagg aagctggccc agaagctggg tgtggagcag cctaccctgt
     2821 actggcatgt gaagaacaag agggctctgc ttgatgccct ggccattgag atgttggaca
     2881 ggcaccacac ccacttctgc cctctggaag gggagtcctg gcaggacttc ctgaggaaca
     2941 atgccaagag cttcagatgt gccttgctct cccaccggga cggtgccaaa gttcacttgg
     3001 gcaccaggcc tacagagaag cagtatgaga ccctggagaa ccagctggca ttcctgtgcc
     3061 aacaaggctt ctccctggaa aatgccttgt atgccctctc tgctgtgggc cacttcacct
     3121 tgggctgtgt gctggaggac caggagcacc aagttgccaa ggaggagagg gagaccccca
     3181 ccactgactc catgccacca ctgctgcggc aagctattga gttgtttgac caccaagggg
     3241 ctgagcctgc attcctgttt ggcctggaac tgatcatctg tggcctggaa aagcagctga
     3301 aatgtgagtc tggctctttc gaagagggca gaggaagcct tctaacatgc ggtgacgtgg
     3361 aggagaatcc cggcccttcc ggaatggtgt ctaagggcga agagctgatt aaggagaaca
     3421 tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag tgcacatccg
     3481 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     3541 gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc agcagaacct
     3601 tcatcaacca cacccagggc atccccgact tctttaagca gtccttccct gagggcttca
     3661 catgggagag agtcaccaca tacgaggacg ggggcgtgct gaccgctacc caggacacca
     3721 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcccatcca
     3781 acggccctgt gatgcagaag aaaacactcg gctgggaggc caacaccgag atgctgtacc
     3841 ccgctgacgg cggcctggaa ggcagaagcg acatggccct gaagctcgtg ggcgggggcc
     3901 acctgatctg caacttcaag accacataca gatccaagaa acccgctaag aacctcaaga
     3961 tgcccggcgt ctactatgtg gaccacagac tggaaagaat caaggaggcc gacaaagaga
     4021 cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct agcaaactgg
     4081 ggcacaaact taatgctagc accggtagat ccgagggcag aggaagcctt ctaacatgcg
     4141 gtgacgtgga ggagaatccc ggcccttccg ggatgaccga gtacaagccc acggtgcgcc
     4201 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     4261 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     4321 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     4381 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     4441 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     4501 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     4561 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     4621 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     4681 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     4741 cctggtgcat gacccgcaag cccggtgcct gaatctaggt cgacaatcaa cctctggatt
     4801 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     4861 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     4921 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     4981 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     5041 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     5101 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     5161 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     5221 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     5281 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tctccgcctt cgccctcaga
     5341 cgagtcggat ctctctttgg gccgcctccc cgcctggtac ctttaagacc aatgacttac
     5401 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     5461 cactcccaac gaagataaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     5521 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     5581 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     5641 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt
     5701 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
     5761 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5821 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5881 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     5941 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     6001 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta gacttttgca
     6061 gagaccaaat tcgtaatcat gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     6121 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     6181 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6241 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6301 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6361 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6421 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6481 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6541 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6601 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6661 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6721 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6781 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6841 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6901 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6961 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     7021 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     7081 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7141 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7201 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7261 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7321 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7381 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7441 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7501 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7561 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7621 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7681 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7741 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7801 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7861 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7921 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7981 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     8041 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     8101 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     8161 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8221 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     8281 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     8341 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     8401 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     8461 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     8521 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     8581 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     8641 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc
     8701 aagctg
//
