LOCUS Exported 6500 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Vector for Flp-dependent expression of EGFP tagged KASH protein domain. ACCESSION . VERSION . KEYWORDS pAAV-FLEXfrt-EGFP-KASH SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6500) AUTHORS Hunker AC, Soden ME, Krayushkina D, Heymann G, Awatramani R, Zweifel LS TITLE Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient Mutagenesis in the Adult Mouse Nervous System. JOURNAL Cell Rep. 2020 Mar 24;30(12):4303-4316.e6. doi: 10.1016/j.celrep.2020.02.092. PUBMED 32209486 REFERENCE 2 (bases 1 to 6500) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6500 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR primer_bind 957..979 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1045..1064 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind 1097..1130 /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)" CDS complement(1448..2164) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS complement(1448..2164) /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1490..1511) /label=EGFP-C /note="EGFP, forward primer" primer_bind 1839..1858 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2098..2119 /label=EGFP-N /note="EGFP, reverse primer" misc_feature 2320..2908 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2373..2393) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2791..2802) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(2935..2952) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2941..3165 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter complement(3375..3704) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3389..3524 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(3443..3462) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind 3689..3709 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" primer_bind complement(3779..3796) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3950..4538) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4030..4049) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4709..5569) /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 5332..5351 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5570..5674) /gene="bla" /label=AmpR promoter primer_bind 5742..5760 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(5798..5820) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 5920..5939 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6153..6172 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 6462..6480 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 6462..6479 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 181 aggtaccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 241 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 301 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 361 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 421 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc 481 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg 541 cggggggggg gggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc 601 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag 661 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc 721 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg 781 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct 841 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc 901 ggctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt 961 cggcttctgg cgtgtgaccg gcggctctag ctagagcctc tgctaaccat gttcatgcct 1021 tcttcttttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg 1081 gcaaagaatt ggatccgaag ttcctattct ctagaaagta taggaacttc caaggcctga 1141 agttcctatt cttcaaaagg tataggaact tcgcggccgc ctaggtggga ggtggcccgt 1201 tggtgtaccg cagcatgggg tagaaggatc gggcaaagtt gttggcctgg gtgcagctgt 1261 agtcatcttc agaggcaggt agcaggcagg ccaggagcag cagcagcagc agaagcagct 1321 gcaacggtag cgctgccctg atcacccttg agaggaagga gcgtctcggc tgggagctgc 1381 cggggctgtc gaggtggggc atcctgctgt ctgtctcctc ctcctcctct cgagatctga 1441 gtccggactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca 1501 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca 1561 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt 1621 ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga 1681 tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca 1741 gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt 1801 tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct 1861 tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt 1921 gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca 1981 cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct 2041 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt 2101 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca 2161 ccatggcgcg ccgaccggtt tagatcttta ctagtttaag cttcaattgg aagttcctat 2221 tctttctaga gtataggaac ttcctgcagt aggctagcga agttcctatt ccttttgaag 2281 tataggaact tcgtcgaggg ggggcccgag cttatcgata atcaacctct ggattacaaa 2341 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac 2401 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc 2461 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt 2521 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc 2581 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc 2641 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg 2701 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt 2761 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc 2821 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt 2881 cggatctccc tttgggccgc ctccccgcat cgataccgtc gactcgctga tcagcctcga 2941 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 3001 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 3061 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 3121 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 3181 gaaccagctg gggctcgact agagcatggc tacgtagata agtagcatgg cgggttaatc 3241 attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 3301 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg 3361 agcgcgcaga gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg 3421 aatagctcag aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca 3481 tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc 3541 gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag 3601 cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg 3661 cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca cagctgcatt 3721 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 3781 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 3841 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 3901 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 3961 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 4021 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 4081 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 4141 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 4201 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 4261 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 4321 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 4381 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 4441 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 4501 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 4561 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 4621 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 4681 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 4741 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4801 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 4861 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 4921 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 4981 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 5041 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 5101 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 5161 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 5221 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 5281 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 5341 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 5401 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 5461 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 5521 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 5581 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 5641 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5701 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 5761 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 5821 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 5881 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac 5941 tgagagtgca ccattcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt 6001 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg 6061 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg 6121 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca 6181 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctggcta 6241 gcgatgaccc tgctgattgg ttcgctgacc atttccgggt gcgggacggc gttaccagaa 6301 actcagaagg ttcgtccaac caaaccgact ctgacggcag tttacgagag agatgatagg 6361 gtctgcttca gtaagccaga tgctacacaa ttaggcttgt acatattgtc gttagaacgc 6421 ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg acactataga 6481 atacacggaa ttaattctag //