LOCUS Exported 8050 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Exchange cassette containing inducible TauP301L 2N4R-EGFP fusion, CAG-driven rtTA3, Puromycin resistance marker, targets AAVS1 site. ACCESSION . VERSION . KEYWORDS AAVS1 CAG rtTA3 TauP301L 2N4R-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8050) AUTHORS Wang X, Friesen E, Muller I, Lemieux M, Dukart R, Maia IB, Kalia S,, Schmitt-Ulms G TITLE Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies. JOURNAL Bio-protocol. 2020; 10(9): e3615 REFERENCE 2 (bases 1 to 8050) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8050 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 23..611 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 512..531 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 765..782 /label=L4440 /note="L4440 vector, forward primer" protein_bind 990..1023 /label=lox66 /bound_moiety="Cre recombinase" /note="Right element (RE) mutant of loxP (Araki et al., 2010). Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 1026..1059 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" misc_feature 1064..1089 /label=SA /note="splice acceptor site" CDS 1113..1166 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 1176..1775 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(1176..1195) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1672..1692 /label=Puro-F /note="Puromycin resistance gene, forward primer" protein_bind 1778..1811 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" polyA_signal 1814..2038 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" polyA_signal 2045..2126 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" CDS complement(2264..2971) /codon_start=1 /product="reverse tetracycline transactivator 3" /label=rtTA3 /note="activates expression from Tet-responsive promoters in the presence of doxycycline" /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPG" primer_bind complement(3015..3034) /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" primer_bind complement(3096..3118) /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" promoter complement(3397..3674) /label=chicken beta-actin promoter polyA_signal 3941..4062 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3952..3971) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(4185..4904) /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS complement(4188..4904) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4230..4251) /label=EGFP-C /note="EGFP, forward primer" primer_bind 4579..4598 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4838..4859 /label=EGFP-N /note="EGFP, reverse primer" regulatory 4901..4910 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 6278..6287 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(6319..6343) /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter complement(6360..6674) /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" primer_bind complement(6363..6387) /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 6434..6452 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6470..6488 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6505..6523 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6541..6559 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6577..6595 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6612..6630 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6648..6666 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6691..6724 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." CDS complement(7089..7949) /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 7712..7731 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 61 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 661 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 721 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 781 aggaactgtc atggcatctt ccaggggtcc gagagctcag ctagtcttct tcctccaacc 841 cgggccccta tgtccacttc aggacagcat gtttgctgcc tccagggatc ctgtgtcccc 901 gagctgggac caccttatat tcccagggcc ggttaatgtg gctctggttc tgggtacttt 961 tatctgtccc ctccacccca cagtggggca taacttcgta tagcatacat tatacgaacg 1021 gtaaagaagt tcctattctc tagaaagtat aggaacttcg cttctgacct cttctcttcc 1081 tcccacaggg cctcgagaga tctggcagcg gagagggcag aggaagtctt ctaacatgcg 1141 gtgacgtgga ggagaatccc ggccctaggc tcgagatgac cgagtacaag cccacggtgc 1201 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg 1261 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc 1321 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg 1381 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg 1441 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga 1501 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg 1561 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg 1621 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc 1681 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc 1741 gcacctggtg catgacccgc aagcccggtg cctgatcgaa gttcctattc tctagaaagt 1801 ataggaactt cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 1861 ccttccttga ccctggaaga tgccactccc actgtccttt cctaataaaa tgaggaaatt 1921 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 1981 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgggt 2041 cgaggcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 2101 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 2161 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg 2221 ctgattatga tcctctagag atatcgtcga caagcttatc gatttacccg gggagcatgt 2281 caaggtcaaa atcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg 2341 cgtcggtcgg cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt 2401 ccaggccgaa aaggaaggca ggttcggctc cctgccggtc gaacagctca attgcttgtc 2461 tcagaagtgg gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat 2521 gctcctgttc ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca 2581 gtgcgttctc cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt 2641 cgtactgttt ctctgttggg cgggtgccga gatgcacttt agccccgtcg cgatgtgaga 2701 ggagagcaca gcggtatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt 2761 ccagggggca gaagtgggta tgatgcctgt ccagcatctc gattggcagg gcatcgagca 2821 gggcccgctt gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag 2881 cgagtttcct tgtcgtcagg ccttcgatac cgacaccatt gagtaattcc agagctccgt 2941 ttatgacttt gctcttgtcc agcctagaca tggtggcact agtgatccaa ttctttgcca 3001 aaatgatgag acagcacaat aaccagcacg ttgcccagga gctgtaggaa aaagaagaag 3061 gcatgaacat ggttagcaga ggctctagag ccgccggtca cacgccagaa gccgaacccc 3121 gccctgcccc gtcccccccg aaggcagccg tccccccgcg gacagccccg agccgctccc 3181 cccgcacaaa gggccctccc ggagcccctc aaggctttca cgcagccaca gaaaagaaac 3241 aagccgtcat taaaccaagc gctaattaca gcccggagga gaagggccgt cccgcccgct 3301 cacctgtggg agtaacgcgg tcagtcagag ccggggcggg cggcgcgagg cggcggcgga 3361 gcggggcacg gggcgaaggc agcgcgcagc gactcccgcc cgccgcgcgc ttcgcttttt 3421 atagggccgc cgccgccgcc gcctcgccat aaaaggaaac tttcggagcg cgccgctctg 3481 attggctgcc gccgcacctc tccgccycsc cccsccccsc cccccscccc sccccscccc 3541 sccyggcgcg cgcccccccc ccccccccgc ccccatcgct gcacaaaata attaaaaaat 3601 aaataaatac aaaattgggg gtggggaggg gggggagatg gggagagtga agcagaacgt 3661 ggggctcacc tcgaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg 3721 aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac 3781 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta 3841 ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac 3901 atacgtcatt attgacgtca atggtacgat cttcctagct taagatacat tgatgagttt 3961 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct 4021 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt 4081 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc 4141 tacaaatgtg gtatggctga ttatgatcta gagtcgcggc cgctttactt gtacagctcg 4201 tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc 4261 ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt gtcgggcagc 4321 agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg 4381 ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg 4441 gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg 4501 ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg 4561 aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc 4621 tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg 4681 tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg 4741 ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc 4801 tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg 4861 atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatggtggc gaccggtgga 4921 tcccgggccc gcggtaccgt cgactgcaga attcttatca aaccctgctt ggccagggag 4981 gcagacacct cgtcagctag cgtggcgagc tggggcgagt ctaccatgtc gatgctgccg 5041 gtggaggaga cattgctgag atgccgtgga gacgtgtccc cagacaccac tggcgacttg 5101 tacacgatct ccgccccgtg gtctgtcttg gctttggcgt tctcgcggaa ggtcagcttg 5161 tgggtttcaa tctttttatt tcctccgcca gggacgtggg tgatattgtc cagggaccca 5221 atcttcgact ggactctgtc cttgaagtca agcttctcag attttacttc cacctggcca 5281 cctcctggtt tatgatggat gttgcctaat gagccacact tggaggtcac cttgctcagg 5341 tcaactggtt tgtagactat ttgcacactg ccgcctccca ggacgtgttt gatattatcc 5401 tttgagccac acttggactg gacgttgcta agatccagct tcttattaat tatctgcacc 5461 ttcccgcctc ccggctggtg cttcaggttc tcagtggagc cgatcttgga cttgacattc 5521 ttcaggtctg gcatgggcac gggggctgtc tgcaggcggc tcttggcgga agacggcgac 5581 ttgggtggag tacggaccac tgccaccttc ttgggctccc gggtgggtgg ggttggaagg 5641 gacggggtgc gggagcggct gccgggagtg cctggggagc cggggctgct gtagccgctg 5701 cgatcccctg attttggagg ttcaccagag ctgggtggtg tctttggagc gggcggggtt 5761 tttgctggaa tcctggtggc gttggcctgg cccttctggc ctggaggggc tgctccccgc 5821 ggtgtggcga tcttcgtttt accatcagcc cccttggctt ttttgtcatc gcttccagtc 5881 ccgtctttgc ttttactgac catgcgagct tgggtcacgt gaccagcagc ttcgtcttcc 5941 aggctggggg tgtctccaat gcctgcttct tcagctgtgg ttccttctgg gatctccgtg 6001 tggggctgcg cggcagcctg cttgccggga gctccctcat ccactaaggg tgctgtcaca 6061 tcttccgctg ttggagtgct cttagcatca gaggtttcag agcccggttc ctcagatccg 6121 tcctcagtgg gggtctgcag gggagattct ttcaggccag cgtccgtgtc accctcttgg 6181 tcttggtgca tggtgtagcc cccctgatct ttcctgtccc ccaacccgta cgtcccagcg 6241 tgatcttcca tcacttcgaa ctcctggcgg ggctcagcca tggtggcgct gaggaccggt 6301 ccaacctgaa aaccgcggag cctgcttttt tgtacaaact tgttgatcgg aattctccag 6361 gcgatctgac ggttcactaa acgagctctg cttatatagg cctcccaccg tacacgccta 6421 cctcgacata cgttctctat cactgatagg gagtaaactc gacatacgtt ctctatcact 6481 gatagggata aactcgacat acgttctcta tcactgatag ggagtaaact cgacatacgt 6541 tctctatcac tgatagggag taaactcgac atacgttctc tatcactgat agggagtaaa 6601 ctcgacatcg ttctctatca ctgataggga gtaaactcga catacgttct ctatcactga 6661 tagggagtaa actcgacagt actaagcttt ataacttcgt ataggatact ttatacgaag 6721 ttatactagg gacaggattg gtgacagaaa agccccatcc ttaggcctcc tccttcctag 6781 tctcctgata ttgggtctaa cccccacctc ctgttaggca gattccttat ctggtgacac 6841 acccccattt cctggagcca tctctctcct tgccagaacc tctaaggttt gcttacgatg 6901 gagccagcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 6961 ttctaaatac attcaaatat gtatccgctc atgagattat caaaaaggat cttcacctag 7021 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 7081 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 7141 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 7201 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 7261 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 7321 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 7381 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 7441 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 7501 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 7561 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 7621 tgcttttctg tgactggtga atactcaacc aagtcattct gagaatagtg tatgcggcga 7681 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 7741 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 7801 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 7861 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 7921 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 7981 tatcagggtt attgtctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 8041 tcagaccccg //