LOCUS       V006881                 9886 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V006881
VERSION     V006881
KEYWORDS    pTet-O-Ngn2-puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9886)
  AUTHORS   Zhang Y, Pak C, Han Y, Ahlenius H, Zhang Z, Chanda S, Marro S,
            Patzke C, Acuna C, Covy J, Xu W, Yang N, Danko T, Chen L, Wernig M,
            Sudhof TC
  TITLE     Rapid single-step induction of functional neurons from human
            pluripotent stem cells.
  JOURNAL   Neuron. 2013 Jun 5;78(5):785-98. doi: 10.1016/j.neuron.2013.05.029.
   PUBMED   23764284
REFERENCE   2  (bases 1 to 9886)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9886)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi:
            "10.1016/j.neuron.2013.05"; journalName: "Neuron"; date: "2013-06-5-
            5"; volume: "78"; issue: "5"; pages: "785-98"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        100..479
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        480..682
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     LTR             697..877
                     /label="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    924..1049
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1542..1775
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             1960..2004
                     /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             2153..2194
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     misc_feature    2302..2419
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     protein_bind    2476..2746
                     /label="tetracycline response element"
                     /note="contains seven copies of the tetracycline operator
                     tetO"
     promoter        2779..2817
                     /label="minimal CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     primer_bind     2814..2838
                     /label="LNCX"
                     /note="Human CMV promoter, forward primer"
     regulatory      2917..2926
                     /label="Kozak sequence"
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2926..2949
                     /label="FLAG"
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             2950..3735
                     /gene="Neurog2"
                     /label="Neurogenin-2"
                     /note="Neurogenin-2 from Mus musculus. Accession#: P70447"
     CDS             3742..3795
                     /label="T2A"
                     /note="2A peptide from Thosea asigna virus capsid protein"
     CDS             3802..4398
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     misc_feature    4440..5028
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     primer_bind     complement(5031..5047)
                     /label="KS primer"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     LTR             5553..5733
                     /label="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    5765..5989
                     /label="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6035..6463
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6477..6806
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     promoter        6854..6901
                     /label="EM7 promoter"
                     /note="synthetic bacterial promoter"
     CDS             6920..7291
                     /label="BleoR"
                     /note="antibiotic-binding protein"
     polyA_signal    7424..7557
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7594..7610)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(7618..7634)
                     /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(7642..7672)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7687..7708)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(7825..7842)
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7996..8584)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(8758..9615)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(9616..9720)
                     /label="AmpR promoter"
     primer_bind     complement(9795..9814)
                     /label="pRS-marker"
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
ORIGIN
        1 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
       61 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
      121 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      181 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      241 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      301 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      361 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      421 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      481 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
      541 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      601 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      661 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      721 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      781 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      841 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      901 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      961 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
     1021 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
     1081 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
     1141 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
     1201 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     1261 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     1321 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     1381 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1441 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1501 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1561 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1621 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1681 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1741 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1801 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1861 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1921 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1981 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     2041 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     2101 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     2161 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     2221 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     2281 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     2341 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     2401 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaga
     2461 tctcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc
     2521 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
     2581 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
     2641 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
     2701 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
     2761 gctcggtacc cgggtcgagg taggcgtgta cggtgggagg cctatataag cagagctcgt
     2821 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
     2881 caccgggacc gatccagcct ccgcggcccc gaattcgcca ccatggacta caaggacgac
     2941 gatgacaagt tcgtcaaatc tgagactctg gagttgaagg aggaagagga ggtactgatg
     3001 ctgctgggct cggcttcccc ggcctcggcg accctgaccc cgatgtcctc cagcgcggac
     3061 gaggaggagg acgaggagct gcgccggccg ggctccgcgc gtgggcagcg tggagcggaa
     3121 gccgggcagg gggtgcaggg cagtccggcg tcgggtgccg ggggttgccg gccagggcgg
     3181 ctgctgggcc tgatgcacga gtgcaagcgt cgcccgtcgc gctcacgggc cgtctcccga
     3241 ggtgccaaga cggcggagac ggtgcagcgc atcaagaaga cccgcaggct caaggccaac
     3301 aaccgcgagc gcaaccgcat gcacaaccta aacgccgcgc tggacgcgct gcgcgaggtg
     3361 ctgcccacct tccccgagga tgccaagctc acgaagatcg agacgctgcg cttcgcccac
     3421 aattacatct gggcgctcac cgagactctg cgcctggcgg accactgcgc cggcgccggt
     3481 ggcctccagg gggcgctctt cacggaggcg gtgctcctga gcccgggagc tgcgctcggc
     3541 gccagcgggg acagcccttc tccaccttcc tcctggagct gcaccaacag cccggcgtca
     3601 tcctccaact ccacgtcccc atacagctgc actttatcgc ccgctagccc cgggtcagac
     3661 gtggactact ggcagccccc acctccggag aagcatcgtt atgcgcctca cctgcccctc
     3721 gccagggact gtatctctag agagggcagg ggaagtcttc taacatgcgg ggacgtggag
     3781 gaaaatcccg gcccactcga gatgaccgag tacaagccca cggtgcgcct cgccacccgc
     3841 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     3901 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     3961 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     4021 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     4081 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     4141 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     4201 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     4261 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     4321 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     4381 acccgcaagc ccggtgcctg attaattaag ctagctaatt cgatatcaag cttatcgata
     4441 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     4501 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     4561 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     4621 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     4681 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     4741 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     4801 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     4861 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     4921 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     4981 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
     5041 gacctcgaga cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg
     5101 ctgattgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc
     5161 aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     5221 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt
     5281 ggatctacca cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga
     5341 tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg
     5401 tagaagaagc caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga
     5461 tggatgaccc ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc
     5521 acatggcccg agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag
     5581 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     5641 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     5701 gaccctttta gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc
     5761 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     5821 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     5881 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     5941 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     6001 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     6061 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     6121 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     6181 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     6241 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     6301 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     6361 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     6421 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     6481 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     6541 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     6601 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     6661 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     6721 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     6781 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     6841 ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     6901 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     6961 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     7021 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     7081 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     7141 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     7201 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     7261 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac
     7321 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     7381 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     7441 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     7501 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     7561 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     7621 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     7681 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     7741 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     7801 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     7861 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     7921 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     7981 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     8041 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     8101 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     8161 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     8221 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     8281 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     8341 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     8401 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     8461 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     8521 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     8581 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     8641 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     8701 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     8761 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     8821 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     8881 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     8941 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     9001 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     9061 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     9121 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     9181 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     9241 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     9301 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     9361 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     9421 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     9481 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     9541 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     9601 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     9661 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     9721 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     9781 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     9841 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaag
//
