LOCUS Exported 5520 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION An AAV genome with tet-inducible, Cre-dependent expression of the fluorescent protein eYFP with a C-terminal Hras farnesylation sequence. ACCESSION . VERSION . KEYWORDS pAAV-TRE-DIO-eYFP-f SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5520) AUTHORS Challis RC, Ravindra Kumar S, Chan KY, Challis C, Beadle K, Jang MJ, Kim HM, Rajendran PS, Tompkins JD, Shivkumar K, Deverman BE, Gradinaru V TITLE Systemic AAV vectors for widespread and targeted gene delivery in rodents. JOURNAL Nat Protoc. 2019 Feb;14(2):379-414. doi: 10.1038/s41596-018-0097-3. PUBMED 30626963 REFERENCE 2 (bases 1 to 5520) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5520 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" protein_bind 165..183 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 202..220 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 239..257 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 276..294 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 313..331 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 350..368 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 387..405 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 424..442 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 464..488 /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 587..620 /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)." protein_bind 671..704 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(776..1492) /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(818..839) /label=EGFP-C /note="EGFP, forward primer" primer_bind 1167..1186 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1426..1447 /label=EGFP-N /note="EGFP, reverse primer" regulatory 1489..1498 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(1505..1538) /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)." protein_bind complement(1589..1622) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 1647..2235 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1700..1720) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2118..2129) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2267..2743 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2466..2485) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 2783..2923 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 2783..2912 /label=AAV2 ITR rep_origin 2998..3453 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3085..3104) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3295..3316 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3470..3489) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3589..3611 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3649..3667) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3735..3839 /gene="bla" /label=AmpR promoter CDS 3840..4700 /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 complement(4058..4077) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4871..5459 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5360..5379 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggccgcac gcgtatcaag tgccacctga cgtctcccta tcagtgatag 181 agaagtcgac acgtctcgag ctccctatca gtgatagaga aggtacgtct agaacgtctc 241 cctatcagtg atagagaagt cgacacgtct cgagctccct atcagtgata gagaaggtac 301 gtctagaacg tctccctatc agtgatagag aagtcgacac gtctcgagct ccctatcagt 361 gatagagaag gtacgtctag aacgtctccc tatcagtgat agagaagtcg acacgtctcg 421 agctccctat cagtgataga gaagctaccc cctatataag cagagctcgt ttagtgaacc 481 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc 541 gatccagcct ggatccggta ccggatcctc tagagtcgac tccggaataa cttcgtatag 601 gatactttat acgaagttat gcagaatggt agctggattg tagctgctat tagcaatatg 661 aaacctctta ataacttcgt atagcataca ttatacgaag ttatggcgcg ccttaggaga 721 gcacacactt gcagctcatg cagccggggc cactctcatc aggagggttc agcttcttgt 781 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt 841 gatcgcgctt ctcgttgggg tctttgctca gggcggactg gtagctcagg tagtggttgt 901 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct 961 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct 1021 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca 1081 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt 1141 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg 1201 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt 1261 ggtcggggta gcgggcgaag cactgcaggc cgtagccgaa ggtggtcacg agggtgggcc 1321 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg 1381 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct 1441 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggcgc 1501 tagcataact tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc 1561 actgttattc tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt 1621 atgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat 1681 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc 1741 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct 1801 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca 1861 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt 1921 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg 1981 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga 2041 aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt 2101 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc 2161 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt 2221 gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg 2281 tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct 2341 tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat 2401 ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg 2461 cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc 2521 cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc 2581 atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt 2641 ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt 2701 gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac 2761 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg 2821 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc 2881 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat 2941 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg 3001 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 3061 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 3121 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 3181 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat 3241 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 3301 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag 3361 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg 3421 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct 3481 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 3541 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 3601 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 3661 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 3721 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 3781 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 3841 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 3901 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 3961 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 4021 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 4081 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 4141 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 4201 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 4261 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 4321 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 4381 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 4441 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 4501 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 4561 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 4621 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 4681 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 4741 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 4801 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 4861 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 4921 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 4981 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 5041 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 5101 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 5161 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 5221 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 5281 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 5341 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 5401 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 5461 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt //