LOCUS       V010363                 8676 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V010363
VERSION     V010363
KEYWORDS    pRSIT17-U6Tet-sh-HTS6-CMV-TetRep-2A-TagGFP2-2A-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8676)
  AUTHORS   Cellecta
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8676)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8676
                     /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..4064
                     /label="TagGFP2"
                     /note="monomeric green fluorescent protein, also known as
                     mTagGFP"
     CDS             4083..4136
                     /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             4143..4739
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     misc_feature    4755..5343
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             5417..5650
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5722..5856
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      5883..6018
                     /label="SV40 ori"
                     /note="SV40 origin of replication"
     primer_bind     complement(6051..6067)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(6075..6091)
                     /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(6099..6129)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6144..6165)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6453..7041)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(7215..8072)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(8073..8177)
                     /label="AmpR promoter"
     primer_bind     8651..8667
                     /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 atgagcgggg
     3361 gcgaggagct gttcgccggc atcgtgcccg tgctgatcga gctggacggc gacgtgcacg
     3421 gccacaagtt cagcgtgcgc ggcgagggcg agggcgacgc cgactacggc aagctggaga
     3481 tcaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctg gtgaccaccc
     3541 tctgctacgg catccagtgc ttcgcccgct accccgagca catgaagatg aacgacttct
     3601 tcaagagcgc catgcccgag ggctacatcc aggagcgcac catccagttc caggacgacg
     3661 gcaagtacaa gacccgcggc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3721 agctgaaggg caaggacttc aaggaggacg gcaacatcct gggccacaag ctggagtaca
     3781 gcttcaacag ccacaacgtg tacatccgcc ccgacaaggc caacaacggc ctggaggcta
     3841 acttcaagac ccgccacaac atcgagggcg gcggcgtgca gctggccgac cactaccaga
     3901 ccaacgtgcc cctgggcgac ggccccgtgc tgatccccat caaccactac ctgagcactc
     3961 agaccaagat cagcaaggac cgcaacgagg cccgcgacca catggtgctc ctggagtcct
     4021 tcagcgcctg ctgccacacc cacggcatgg acgagctgta cagggctagc accggtagat
     4081 ccgagggcag aggaagcctt ctaacatgcg gtgacgtgga ggagaatccc ggcccttccg
     4141 ggatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     4201 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     4261 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     4321 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     4381 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     4441 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     4501 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     4561 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     4621 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     4681 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     4741 gaatctaggt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     4801 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     4861 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     4921 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     4981 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     5041 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     5101 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     5161 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     5221 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     5281 ctcttccgcg tctccgcctt cgccctcaga cgagtcggat ctctctttgg gccgcctccc
     5341 cgcctggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     5401 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagataaga tctgcttttt
     5461 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     5521 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     5581 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     5641 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     5701 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     5761 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5821 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     5881 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5941 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     6001 gaggcttttt tggaggccta gacttttgca gagaccaaat tcgtaatcat gtcatagctg
     6061 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     6121 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     6181 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6241 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6301 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6361 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6421 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6481 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6541 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6601 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6661 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6721 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6781 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6841 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6901 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6961 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     7021 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     7081 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     7141 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     7201 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     7261 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7321 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7381 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7441 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7501 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7561 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7621 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7681 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7741 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7801 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7861 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7921 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7981 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     8041 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     8101 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     8161 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     8221 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     8281 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     8341 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     8401 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     8461 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca
     8521 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     8581 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     8641 tcacgacgtt gtaaaacgac ggccagtgcc aagctg
//
