LOCUS Exported 7762 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Double floxed Gi-coupled hM4D DREADD fused with mCherry under the control of EF1a promoter . ACCESSION . VERSION . KEYWORDS pAAV-EF1a-DIO-hM4D(Gi)-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7762) AUTHORS Bryan Roth TITLE Roth lab DREADDs JOURNAL Unpublished REFERENCE 2 (bases 1 to 7762) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7762 /organism="synthetic DNA construct" /mol_type="other DNA" regulatory 599..608 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 605..1315 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(750..768) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1018..1037 /label=mCherry-F /note="mCherry, forward primer" protein_bind complement(1324..1357) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(1408..1441) /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)." promoter complement(1472..2650) /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 1481..2419 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind complement(1504..1524) /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" repeat_region 2751..2880 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" rep_origin complement(2942..3530) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3022..3041) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3701..4561) /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 4324..4343 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4562..4666) /gene="bla" /label=AmpR promoter primer_bind 4734..4752 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4790..4812) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4912..4931 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(4948..5403) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5085..5106) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5297..5316 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 5478..5618 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5489..5618 /label=AAV2 ITR polyA_signal 5658..6134 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind 5916..5935 /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" misc_feature 6166..6754 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" CDS 6272..6283 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind 6681..6701 /label=WPRE-R /note="WPRE, reverse primer" protein_bind 6779..6812 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 6863..6896 /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)." regulatory 6903..6912 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 ccccgtggct gataaggaca cttccaatga gtccagctca ggcagtgcca cccagaacac 61 caaggaacgc ccagccacag agctgtccac cacagaggcc accacgcccg ccatgcccgc 121 ccctcccctg cagccgcggg ccctcaaccc agcctccaga tggtccaaga tccagattgt 181 gacgaagcag acaggcaatg agtgtgtgac agccattgag attgtgcctg ccacgccggc 241 tggcatgcgc cctgcggcca acgtggcccg caagttcgcc agcatcgctc gcaaccaggt 301 gcgcaagaag cggcagatgg cggcccggga gcgcaaagtg acacgaacga tctttgccat 361 tctgctggcc ttcatcctca cctggacgcc ctacaacgtc atggtcctgg tgaacacctt 421 ctgccagagc tgcatccctg acacggtgtg gtccattggc tactggctct gctacgtcaa 481 cagcaccatc aaccctgcct gctatgctct gtgcaacgcc acctttaaaa agaccttccg 541 gcacctgctg ctgtgccagt atcggaacat cggcactgcc aggcgggatc caccggtcgc 601 caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt 661 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga 721 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct 781 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa 841 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga 901 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca 961 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc 1021 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga 1081 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga 1141 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa 1201 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta 1261 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaggcgc 1321 gccataactt cgtataatgt atgctatacg aagttattaa gaggtttcat attgctaata 1381 gcagctacaa tccagctacc attctgcata acttcgtata aagtatccta tacgaagtta 1441 ttccggagtc gactctagag gatccggtac ctcacgacac ctgaaatgga agaaaaaaac 1501 tttgaaccac tgtctgaggc ttgagaatga accaagatcc aaactcaaaa agggcaaatt 1561 ccaaggagaa ttacatcaag tgccaagctg gcctaacttc agtctccacc cactcagtgt 1621 ggggaaactc catcgcataa aacccctccc cccaacctaa agacgacgta ctccaaaagc 1681 tcgagaacta atcgaggtgc ctggacggcg cccggtactc cgtggagtca catgaagcga 1741 cggctgagga cggaaaggcc cttttccttt gtgtgggtga ctcacccgcc cgctctcccg 1801 agcgccgcgt cctccatttt gagctccctg cagcagggcc gggaagcggc catctttccg 1861 ctcacgcaac tggtgccgac cgggccagcc ttgccgccca gggcggggcg atacacggcg 1921 gcgcgaggcc aggcaccaga gcaggccggc cagcttgaga ctacccccgt ccgattctcg 1981 gtggccgcgc tcgcaggccc cgcctcgccg aacatgtgcg ctgggacgca cgggccccgt 2041 cgccgcccgc ggccccaaaa accgaaatac cagtgtgcag atcttggccc gcatttacaa 2101 gactatcttg ccagaaaaaa agcgtcgcag caggtcatca aaaattttaa atggctagag 2161 acttatcgaa agcagcgaga caggcgcgaa ggtgccacca gattcgcacg cggcggcccc 2221 agcgcccagg ccaggcctca actcaagcac gaggcgaagg ggctccttaa gcgcaaggcc 2281 tcgaactctc ccacccactt ccaacccgaa gctcgggatc aagaatcacg tactgcagcc 2341 aggtggaagt aattcaaggc acgcaagggc cataacccgt aaagaggcca ggcccgcggg 2401 aaccacacac ggcacttacc tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca 2461 cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg cggagccagt 2521 acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg gttcaattgc 2581 cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc cactgacggg 2641 caccggagcc aattcccact cctttcaaga cctagaaggt ccattagctg caaagattcc 2701 tctctgttta aaactttatc catctttgca aagcttacgc gtgcggccgc aggaacccct 2761 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc 2821 aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg 2881 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 2941 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 3001 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 3061 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 3121 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 3181 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 3241 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 3301 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 3361 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 3421 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 3481 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 3541 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 3601 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 3661 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 3721 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 3781 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 3841 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 3901 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 3961 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 4021 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 4081 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 4141 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 4201 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 4261 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 4321 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 4381 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 4441 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 4501 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 4561 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 4621 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 4681 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 4741 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 4801 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 4861 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 4921 agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg 4981 cgttaaattt ttgttaaatc agctcatttt ttaaccaata gaccgaaatc ggcaaaatcc 5041 cttataaatc aaaagaatag cccgagatag agttgagtgt tgttccagtt tggaacaaga 5101 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 5161 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag 5221 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 5281 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctaaggcg ctggcaagtg 5341 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 5401 cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa 5461 ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg 5521 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 5581 cagagaggga gtggccaact ccatcactag gggttcctgc ggccgctcgg tccgcacgtg 5641 gttacctaca aaatcagaag gacagggaag ggagcagtgg ttcacgcctg taatcccagc 5701 aatttgggag gccaaggtgg gtagatcacc tgagattagg agttggagac cagcctggcc 5761 aatatggtga aaccccgtct ctaccaaaaa aacaaaaatt agctgagcct ggtcatgcat 5821 gcctggaatc ccaacaactc gggaggctga ggcaggagaa tcgcttgaac ccaggaggcg 5881 gagattgcag tgagccaaga ttgtgccact gcactccagc ttggttccca atagaccccg 5941 caggccctac aggttgtctt cccaacttgc cccttgctcc ataccacccc cctccacccc 6001 ataatattat agaaggacac ctagtcagac aaaatgatgc aacttaattt tattaggaca 6061 aggctggtgg gcactggagt ggcaacttcc agggccagga gaggcactgg ggaggggtca 6121 cagggatgcc acccgtagat ctctcgagca gcgctcggta tcgatgcggg gaggcggccc 6181 aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg 6241 gcagcaggcc gcgggaagga aggtccgctg gattgagggc cgaagggacg tagcagaagg 6301 acgtcccgcg cagaatccag gtggcaacat aggcgagcag ccaaggaaag gacgatgatt 6361 tccccgacaa caccacggaa ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg 6421 caaggcaggc ggcgatgagt tccgccgtgg caatagggag ggggaaagcg aaagtcccgg 6481 aaaggagctg acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag 6541 tgcacaccac gccacgttgc ctgacaacgg gccacaactc ctcataaaga gacagcaacc 6601 aggatttata caaggaggag aaaatgaaag ccatacggga agcaatagca tgatacaaag 6661 gcattaaagc agcgtatcca catagcgtaa aaggagcaac atagttaaga ataccagtca 6721 atctttcaca aattttgtaa tccagaggtt gattatcgat aagcttgata tcgaattcat 6781 aacttcgtat agcatacatt atacgaagtt attctttgca ccattctaaa gaataacagt 6841 gataatttct gggttaaggc aaataacttc gtataggata ctttatacga agttatgcta 6901 gcgccaccat ggccaacttc acacctgtca atggcagctc gggcaatcag tccgtgcgcc 6961 tggtcacgtc atcatcccac aatcgctatg agacggtgga aatggtcttc attgccacag 7021 tgacaggctc cctgagcctg gtgactgtcg tgggcaacat cctggtgatg ctgtccatca 7081 aggtcaacag gcagctgcag acagtcaaca actacttcct cttcagcctg gcgtgtgctg 7141 atctcatcat aggcgccttc tccatgaacc tctacaccgt gtacatcatc aagggctact 7201 ggcccctggg cgccgtggtc tgcgacctgt ggctggccct ggactgcgtg gtgagcaacg 7261 cctccgtcat gaaccttctc atcatcagct ttgaccgcta cttctgcgtc accaagcctc 7321 tcacctaccc tgcccggcgc accaccaaga tggcaggcct catgattgct gctgcctggg 7381 tactgtcctt cgtgctctgg gcgcctgcca tcttgttctg gcagtttgtg gtgggtaagc 7441 ggacggtgcc cgacaaccag tgcttcatcc agttcctgtc caacccagca gtgacctttg 7501 gcacagccat tgctggcttc tacctgcctg tggtcatcat gacggtgctg tacatccaca 7561 tctccctggc cagtcgcagc cgagtccaca agcaccggcc cgagggcccg aaggagaaga 7621 aagccaagac gctggccttc ctcaagagcc cactaatgaa gcagagcgtc aagaagcccc 7681 cgcccgggga ggccgcccgg gaggagctgc gcaatggcaa gctggaggag gcccccccgc 7741 cagcgctgcc accgccaccg cg //