LOCUS Exported 7498 bp ds-DNA circular SYN 24-MAY-2022 DEFINITION Chemogenetics Cre-dependent expression plasmid. ACCESSION . VERSION . KEYWORDS AAV SYN flex PSAM4 GlyR IRES EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7498) AUTHORS Magnus CJ, Lee PH, Bonaventura J, Zemla R, Gomez JL, Ramirez MH, Hu X, Galvan A, Basu J, Michaelides M, Sternson SM TITLE Ultrapotent chemogenetics for research and potential clinical applications. JOURNAL Science. 2019 Mar 14. pii: science.aav5282. doi: 10.1126/science.aav5282. PUBMED 30872534 REFERENCE 2 (bases 1 to 7498) AUTHORS . TITLE Direct Submission JOURNAL Exported May 24, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7498 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 56..185 /label=AAV2 ITR polyA_signal 347..468 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(384..403) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 438..457 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" misc_feature 545..1133 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" CDS 651..662 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind 1060..1080 /label=WPRE-R /note="WPRE, reverse primer" protein_bind complement(1201..1234) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(1305..1338) /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)." misc_feature 2703..3288 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2870..2887) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" CDS 3301..4017 /codon_start=1 /product="enhanced GFP with monomerizing A206K mutation (Zacharias et al., 2002)" /label=mEGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(3343..3364) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3604..3623) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3951..3972 /label=EGFP-C /note="EGFP, forward primer" protein_bind 4030..4063 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 4126..4159 /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)." primer_bind complement(4178..4197) /label=pMT2-F /note="Synthetic intron, forward primer" intron 4180..4312 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" repeat_region 4911..5040 /label=AAV2 ITR rep_origin 5112..5567 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5199..5218) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5409..5430 /label=F1ori-F /note="F1 origin, forward primer" promoter 5593..5697 /gene="bla" /label=AmpR promoter CDS 5698..6558 /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(5916..5935) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6729..7317 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7218..7237 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7471..7488 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg 61 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg 121 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg 181 ttccttgtag ttaatgatta acccgccatg ctacttatct acgtagccat gctctaggaa 241 gatccttatc gattttacca catttgtaga ggttttactt gctttaaaaa acctcccaca 301 tctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc 361 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 421 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgctc 481 gaagcggccg gccgcccggg cgacctagat gcattcgacg gccgcccggg tcgacggtat 541 cgatgcgggg aggcggccca aagggagatc cgactcgtct gagggcgaag gcgaagacgc 601 ggaagaggcc gcagagccgg cagcaggccg cgggaaggaa ggtccgctgg attgagggcc 661 gaagggacgt agcagaagga cgtcccgcgc agaatccagg tggcaacaca ggcgagcagc 721 caaggaaagg acgatgattt ccccgacaac accacggaat tgtcagtgcc caacagccga 781 gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt ccgccgtggc aatagggagg 841 gggaaagcga aagtcccgga aaggagctga caggtggtgg caatgcccca accagtgggg 901 gttgcgtcag caaacacagt gcacaccacg ccacgttgcc tgacaacggg ccacaactcc 961 tcataaagag acagcaacca ggatttatac aaggaggaga aaatgaaagc catacgggaa 1021 gcaatagcat gatacaaagg cattaaagca gcgtatccac atagcgtaaa aggagcaaca 1081 tagttaagaa taccagtcaa tctttcacaa attttgtaat ccagaggttg attcttaaga 1141 gcttcgagtg gatcatctgc aaattcgccc ttttcagtgt ttcgaacctc ttcgagggac 1201 ataacttcgt ataatgtatg ctatacgaag ttatgggtcg atggtgagat ctggactaga 1261 gggtcgatgg tgatgcttgg atcaaaatag gaagaatccc aatgataact tcgtataaag 1321 tatcctatac gaagttatac tagtcatatg ggatccgaat tcgctagcgc caccatgcgc 1381 tgttctccag gcggcgtgtg gctcgccctg gctgcttccc ttctgcacgt tagcctgcag 1441 ggtgagttcc agcgcaaact gtataaggag cttgttaaga attataaccc cctggagcgg 1501 ccggtcgcaa atgattccca gccactgaca gtgtacttca gcctctcctt gctgcagatc 1561 atggacgtgg atgaaaagaa ccaggtgctg accactaata tttggttgca gatgtcctgg 1621 accgatcact acttgcagtg gaatgtgagc gaatacccag gtgtaaagac tgtaagattc 1681 cctgacggcc aaatctggaa accagatatc ctgctgtaca acagcgcaga cgaaaggttt 1741 gatgcaacat ttcacaccaa cgtgggagtc aattcttcag gccactgcct gtacctgccc 1801 cctggaatct tcaagtcctc atgctatatc gacgtccgct ggtttccctt cgacgtccag 1861 cactgcaaac tcaaattcgg gagctggagc tacggcggat ggagcctgga tctgcaaatg 1921 caggaggctg acatctctgg ttacatcccg aatggggagt gggaccttgt gggaatcccc 1981 ggtaaaagaa gcgagcgatt ttatgaatgc tgcaaagagc ccttcccaga tgtcaccttc 2041 acagtgacca tgcggagacg catgggttat tatctgatcc aaatgtatat cccaagcttg 2101 cttatagtga ttttgtcatg gatctccttc tggattaata tggacgccgc tccagctagg 2161 gtcggactgg gcatcaccac agtgctgaca atgactactc agagctcagg cagccgagcc 2221 agcttgccca aggtttctta cgtgaaggcc atcgatatct ggatggctgt ctgccttctg 2281 tttgtcttca gcgcactgct ggaatacgcc gctgtcaatt ttgtgtctcg acagcataaa 2341 gagctgttgc ggttcagaag aaaacgacgc caccacaaag aggatgaggc aggagaagga 2401 cgcttcaact ttagcgccta tggtatggga cctgcttgcc tccaggctaa agacggaatt 2461 tccgtgaagg gagccaacaa tagcaacaca accaacccac cccctgctcc atctaagagc 2521 ccggaggaaa tgcgcaaact ctttattcag agagcgaaaa agatcgacaa aatctcccgg 2581 atcggattcc ccatggcttt cctgattttc aacatgtttt attggatcat ctacaagatt 2641 gtgcgaaggg aggacgtaca caaccagtaa gcggccgcaa ttccccccgc cccccccccc 2701 cccccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 2761 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 2821 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 2881 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 2941 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 3001 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 3061 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctaagc gtattcaaca 3121 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 3181 gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 3241 gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat gggggatccg 3301 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 3361 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 3421 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 3481 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 3541 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 3601 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 3661 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 3721 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 3781 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 3841 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 3901 ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg 3961 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatct 4021 agcaagctta taacttcgta tagcatacat tatacgaagt tatgtgtcga tggtgatgct 4081 tggcaattcg ggtcgatggt gaagcattgg tcttcctatt ttgatataac ttcgtatagg 4141 atactttata cgaagttatg gggggcgcgc catcatcacc tgtggagaga aaggcaaagt 4201 ggatgtcagt aagaccaata ggtgcctatc agaaacgcaa gagtcttctc tgtctcgaca 4261 agcccagttt ctattggtct ccttaaacct gtcttgtaac cttgatactt acctgatcca 4321 acggctgcga cttggggcag ggggtcctag gggtggtctg gccaggagcc gcgggggcgg 4381 actgcgcggt gcccaggagc acagctgcgc tctcaggcac gacacgactc ctccgctgcc 4441 caccgcagac tgaggcagcg ctgagtcgcc ggcgccgcag cgcagatggt cgcgcccgtg 4501 cccccctatc tcgcgcctcg cgtggtgcgg tccggctggg ccggcggcgg cgcggacgcg 4561 accaaggtgg ccgggaaggg gagtttgcgg gggaccggcg agtgacgtca gcgcgccttc 4621 agtgctgagg cggcggtggc gcgcgccgcc aggcgggggc gaaggcactg tccgcggtgc 4681 tgaagctggc agtgcgcacg cgcctcgccg catcctgttt cccctccccc tctctgatag 4741 gggatgcgca atttggggaa tgggggttgg gtgcttgtcc agtgggtcgg ggtcggtcgt 4801 caagcatgct ttaaatactg aaaagggcca attccagcac actggagctc ggatcttcct 4861 agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct 4921 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc 4981 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag 5041 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 5101 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 5161 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 5221 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 5281 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 5341 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 5401 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 5461 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 5521 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt 5581 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 5641 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 5701 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 5761 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 5821 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 5881 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 5941 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 6001 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 6061 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 6121 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 6181 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 6241 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 6301 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 6361 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 6421 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 6481 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 6541 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 6601 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 6661 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 6721 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 6781 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 6841 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc 6901 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 6961 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 7021 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 7081 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 7141 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 7201 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 7261 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 7321 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 7381 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 7441 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaag //