LOCUS Exported 6674 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV plasmid with Ple32 (CLDN5 MiniPromoter) driving expression of EmGFP. Contains WPRE.. ACCESSION . VERSION . KEYWORDS pEMS2181 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6674) AUTHORS Korecki AJ, Cueva-Vargas JL, Fornes O, Agostinone J, Farkas RA, Hickmott JW, Lam SL, Mathelier A, Zhou M, Wasserman WW, Di Polo A, Simpson EM TITLE Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Muller glial, and PAX6 cells. JOURNAL Gene Ther. 2021 Feb 2. pii: 10.1038/s41434-021-00227-z. doi: 10.1038/s41434-021-00227-z. PUBMED 33531684 REFERENCE 2 (bases 1 to 6674) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6674 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR intron 1913..2045 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" primer_bind 2028..2047 /label=pMT2-F /note="Synthetic intron, forward primer" regulatory 2066..2075 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2072..2791 /codon_start=1 /product="Emerald GFP" /label=EmGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2117..2138) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2378..2397) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2725..2746 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(2857..2877) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3275..3286) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3407..3528 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3418..3437) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3472..3491 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 3724..3853 /label=AAV2 ITR primer_bind complement(3872..3889) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3872..3888) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3881..3903) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4030..4485 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4117..4136) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4327..4348 /label=F1ori-F /note="F1 origin, forward primer" promoter 4511..4615 /gene="bla" /label=AmpR promoter CDS 4616..5476 /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(4834..4853) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5647..6235 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6136..6155 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6389..6406 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6523..6544 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6559..6589 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6597..6613 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 6602..6624 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6621..6637 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6621..6637 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 181 aggaagatcg gaattcccta ggccggccct tgccttcaga acctccccac actaggtgag 241 ccagacgctg gccttatctc atttaccatc tcagagccat ctgaaggggg agaagggaac 301 cgggccccag gagggagaaa gtcatcaaac ctcccacatc tgtgaccagc ctcagtgcca 361 tactttttct atggagggcc ctgtccaatg gaactgagca cagaccagat aaaagaactg 421 ggcacccagt ggcctcagtc cagggcctgg agttcaaact ttactggaaa caaaggggcc 481 gagagagact ggggaaagaa ctactagaaa ggggctggtg cccccatggg gctgtgggtt 541 ttggagccgc gtgcccccac ctgagcctca gggggcccgg agtgtccaca ccagtggacc 601 tttcgagaaa tggctgggcc attgtgcaga agaatgcccg gaaatcccgc gcctccctcc 661 tccagcaagg atgggggctc ttcctcctgg ccaggaaact ccaagttggc ttccggaggg 721 tggcctgggg gctggggtgc cagggacacc atcgccactg gtgggagggc agggcacagc 781 ccctccgtgt ccctttgtct ctcctgtctg aaggccagag caggctgcta ggcctggggc 841 caccactgcc cctgggtgct acacccagtg tgctgggtca ctgggaactt cctgaagtgg 901 tgtcacctga actgggcccc caaggatggg gtgcgggcag taccgcagga agaggagcag 961 cccctgtgaa gattgagagg tctgggaagc ccctgcggct tgggagagtg ggggtcgcca 1021 ggcaggggga aagcccctgt gccaccgctt tttgccagag actcaggctc cagagaggca 1081 gtgagtggca tggggggtga ggctggggcc ctgggcctga cctccacacg cctgcctggc 1141 ctctctgttt gccatgggat gagagagaca gtgctgggac tcagagcggg gctggagagt 1201 gagagtgcga gaaagggcct gggtggggct tggaccccgg ggcgggcttt ctggagagcc 1261 cccctacgag ggcctctacg gcggtgacgg ggtggggggc ttctgcaaac cttggtcagg 1321 gaagtggagc tggctcgagt ggaagagacc acccggctca gtcggggatg tgggagtgga 1381 ctgggtggtg cagactgggg gtcgagcgcc ttctgaagtg acggggccgg gacgcgcagg 1441 gaggcggccc aagaagcgcg ccctaggcca gcccagaatg cgctcggccg cgactaggac 1501 aacggcgggt ggggctgggg gcggctgccg ggcggggagc ggtcccgcgc cctcagctac 1561 ccctcaagag ccgttgtttc cctaacttca gctgccagag gctctgtgat tggctgcggc 1621 acgatgaccc gcgcacggat tggctgcttc gggccggggg gccgggcccg ggggacagaa 1681 tccgcccccg aaccttcaaa gagggtaccc cccggcagga gctggcagac ccaggaggtg 1741 cgacagaccc gcggggcaaa cggactgggg ccaagagccg ggagcgcggg cgcaaaggca 1801 ccagggcccg cccagggcgc cgcgcagcac ggccttgggg gttctgcggg ccttcgggtg 1861 cgcgtctcgc ctctagccgg cgcgccgaag ttggtcgtga ggcactgggc aggtaagtat 1921 caaggttaca agacaggttt aaggagacca atagaaactg ggcttgtcga gacagagaag 1981 actcttgcgt ttctgatagg cacctattgg tcttactgac atccactttg cctttctctc 2041 cacaggtgtc cactcccagg cggccgccac catggtgagc aagggcgagg agctgttcac 2101 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 2161 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 2221 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca 2281 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 2341 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 2401 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 2461 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 2521 ggtctatatc accgccgaca agcagaagaa cggcatcaag gtgaacttca agacccgcca 2581 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 2641 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 2701 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 2761 cactctcggc atggacgagc tgtacaagta agcggccgcg atcgctcaac ctctggatta 2821 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg 2881 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc 2941 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca 3001 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac 3061 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact 3121 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc 3181 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg 3241 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc 3301 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac 3361 gagtcggatc tccctttggg ccgcctcccc gcgatcgcag acatgataag atacattgat 3421 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 3481 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat 3541 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa 3601 aacctctaca aatgtggtag tcgaaattcc cgataaggat cttcgtcgac gaattcccga 3661 taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact 3721 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg 3781 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg 3841 agcgagcgcg cagccttaat taacctaatt cactggccgt cgttttacaa cgtcgtgact 3901 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 3961 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 4021 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 4081 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 4141 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 4201 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 4261 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 4321 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 4381 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 4441 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg 4501 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 4561 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 4621 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4681 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4741 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4801 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 4861 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4921 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 4981 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 5041 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 5101 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 5161 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 5221 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5281 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5341 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5401 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5461 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5521 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5581 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5641 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5701 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5761 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 5821 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5881 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 5941 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 6001 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 6061 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 6121 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 6181 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6241 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6301 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6361 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6421 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga 6481 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac 6541 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt 6601 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca gatttaatta 6661 aggccttaat tagg //