LOCUS Exported 8048 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 8048) 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 8048) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8048 /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(3012..3031) /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" primer_bind complement(3093..3115) /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" promoter complement(3395..3672) /label=chicken beta-actin promoter polyA_signal 3939..4060 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3950..3969) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(4183..4902) /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(4186..4902) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4228..4249) /label=EGFP-C /note="EGFP, forward primer" primer_bind 4577..4596 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4836..4857 /label=EGFP-N /note="EGFP, reverse primer" regulatory 4899..4908 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 6276..6285 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(6317..6341) /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter complement(6358..6672) /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" primer_bind complement(6361..6385) /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 6432..6450 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6468..6486 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6503..6521 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6539..6557 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6575..6593 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6610..6628 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6646..6664 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6689..6722 /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(7087..7947) /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 7710..7729 /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 ccctggaagg 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 agtccaattc tttgccaaaa 3001 tgatgagaca gcacaataac cagcacgttg cccaggagct gtaggaaaaa gaagaaggca 3061 tgaacatggt tagcagaggc tctagagccg ccggtcacac gccagaagcc gaaccccgcc 3121 ctgccccgtc ccccccgaag gcagccgtcc ccccgcggac agccccgagc cgctcccccc 3181 gcacaaaggg ccctcccgga gcccctcaag gctttcacgc agccacagaa aagaaacaag 3241 ccgtcattaa accaagcgct aattacagcc cggaggagaa gggccgtccc gcccgctcac 3301 ctgtgggagt aacgcggtca gtcagagccg gggcgggcgg cgcgaggcgg cggcggagcg 3361 gggcacgggg cgaaggcagc gtcgcagcga ctcccgcccg ccgcgcgctt cgctttttat 3421 agggccgccg ccgccgccgc ctcgccataa aaggaaactt tcggagcgcg ccgctctgat 3481 tggctgccgc cgcacctctc cgcctcgccc cgccccgccc ctcgccccgc cccgccccgc 3541 ctggcgcgcg cccccccccc ccccccgccc ccatcgctgc acaaaataat taaaaaataa 3601 ataaatacaa aattgggggt ggggaggggg gggagatggg gagagtgaag cagaacgtgg 3661 ggctcacctc gaccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa 3721 agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt 3781 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc 3841 gtaaatagtc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat 3901 acgtcattat tgacgtcaat ggtacgatct tcctagctta agatacattg atgagtttgg 3961 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 4021 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca 4081 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta 4141 caaatgtggt atggctgatt atgatctaga gtcgcggccg ctttacttgt acagctcgtc 4201 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt 4261 ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag 4321 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc 4381 gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc 4441 catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc 4501 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa 4561 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg 4621 gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta 4681 gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg 4741 cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc 4801 gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat 4861 gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggcga ccggtggatc 4921 ccgggcccgc ggtaccgtcg actgcagaat tcttatcaaa ccctgcttgg ccagggaggc 4981 agacacctcg tcagctagcg tggcgagctg gggcgagtct accatgtcga tgctgccggt 5041 ggaggagaca ttgctgagat gccgtggaga cgtgtcccca gacaccactg gcgacttgta 5101 cacgatctcc gccccgtggt ctgtcttggc tttggcgttc tcgcggaagg tcagcttgtg 5161 ggtttcaatc tttttatttc ctccgccagg gacgtgggtg atattgtcca gggacccaat 5221 cttcgactgg actctgtcct tgaagtcaag cttctcagat tttacttcca cctggccacc 5281 tcctggttta tgatggatgt tgcctaatga gccacacttg gaggtcacct tgctcaggtc 5341 aactggtttg tagactattt gcacactgcc gcctcccagg acgtgtttga tattatcctt 5401 tgagccacac ttggactgga cgttgctaag atccagcttc ttattaatta tctgcacctt 5461 cccgcctccc ggctggtgct tcaggttctc agtggagccg atcttggact tgacattctt 5521 caggtctggc atgggcacgg gggctgtctg caggcggctc ttggcggaag acggcgactt 5581 gggtggagta cggaccactg ccaccttctt gggctcccgg gtgggtgggg ttggaaggga 5641 cggggtgcgg gagcggctgc cgggagtgcc tggggagccg gggctgctgt agccgctgcg 5701 atcccctgat tttggaggtt caccagagct gggtggtgtc tttggagcgg gcggggtttt 5761 tgctggaatc ctggtggcgt tggcctggcc cttctggcct ggaggggctg ctccccgcgg 5821 tgtggcgatc ttcgttttac catcagcccc cttggctttt ttgtcatcgc ttccagtccc 5881 gtctttgctt ttactgacca tgcgagcttg ggtcacgtga ccagcagctt cgtcttccag 5941 gctgggggtg tctccaatgc ctgcttcttc agctgtggtt ccttctggga tctccgtgtg 6001 gggctgcgcg gcagcctgct tgccgggagc tccctcatcc actaagggtg ctgtcacatc 6061 ttccgctgtt ggagtgctct tagcatcaga ggtttcagag cccggttcct cagatccgtc 6121 ctcagtgggg gtctgcaggg gagattcttt caggccagcg tccgtgtcac cctcttggtc 6181 ttggtgcatg gtgtagcccc cctgatcttt cctgtccccc aacccgtacg tcccagcgtg 6241 atcttccatc acttcgaact cctggcgggg ctcagccatg gtggcgctga ggaccggtcc 6301 aacctgaaaa ccgcggagcc tgcttttttg tacaaacttg ttgatcggaa ttctccaggc 6361 gatctgacgg ttcactaaac gagctctgct tatataggcc tcccaccgta cacgcctacc 6421 tcgacatacg ttctctatca ctgataggga gtaaactcga catacgttct ctatcactga 6481 tagggataaa ctcgacatac gttctctatc actgataggg agtaaactcg acatacgttc 6541 tctatcactg atagggagta aactcgacat acgttctcta tcactgatag ggagtaaact 6601 cgacatcgtt ctctatcact gatagggagt aaactcgaca tacgttctct atcactgata 6661 gggagtaaac tcgacagtac taagctttat aacttcgtat aggatacttt atacgaagtt 6721 atactaggga caggattggt gacagaaaag ccccatcctt aggcctcctc cttcctagtc 6781 tcctgatatt gggtctaacc cccacctcct gttaggcaga ttccttatct ggtgacacac 6841 ccccatttcc tggagccatc tctctccttg ccagaacctc taaggtttgc ttacgatgga 6901 gccagcatca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 6961 ctaaatacat tcaaatatgt atccgctcat gagattatca aaaaggatct tcacctagat 7021 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 7081 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 7141 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 7201 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 7261 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 7321 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 7381 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 7441 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 7501 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 7561 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 7621 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 7681 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 7741 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 7801 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 7861 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 7921 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 7981 tcagggttat tgtctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 8041 agaccccg //