LOCUS Exported 5949 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes Cre-dependent short hairpin RNA (shRNA) that targets the 3'-untranslated region of the rat Crh gene which encodes corticotropin releasing factor. ACCESSION . VERSION . KEYWORDS AAV8-hSyn-flex-miR30-eGFP-shCrh SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5949) AUTHORS Pomrenze MB, Giovanetti SM, Maiya R, Gordon AG, Kreeger LJ, Messing RO TITLE Dissecting the Roles of GABA and Neuropeptides from Rat Central Amygdala CRF Neurons in Anxiety and Fear Learning. JOURNAL Cell Rep. 2019 Oct 1;29(1):13-21.e4. doi: 10.1016/j.celrep.2019.08.083. PUBMED 31577943 REFERENCE 2 (bases 1 to 5949) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5949 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..141 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 12..141 /label=AAV2 ITR promoter 186..633 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" protein_bind 671..704 /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 755..788 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." ncRNA complement(825..951) /label=3' miR-30a /note="sequence downstream of the 71-nt precursor of the human miR-30a microRNA (Zeng et al., 2002)" /ncRNA_class="miRNA" ncRNA complement(979..993) /label=miR-30a loop /note="loop from the the 71-nt precursor of the human miR-30a microRNA (Zeng et al., 2002)" /ncRNA_class="miRNA" ncRNA complement(1021..1142) /label=5' miR-30a /note="sequence upstream of the 71-nt precursor of the human miR-30a microRNA (Zeng et al., 2002)" /ncRNA_class="miRNA" CDS complement(1195..1911) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1237..1258) /label=EGFP-C /note="EGFP, forward primer" primer_bind 1586..1605 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1845..1866 /label=EGFP-N /note="EGFP, reverse primer" regulatory 1908..1917 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(1934..1967) /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(2018..2051) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2076..2664 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2129..2149) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2547..2558) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2696..3172 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2895..2914) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 3212..3352 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3212..3341 /label=AAV2 ITR rep_origin 3427..3882 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3514..3533) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3724..3745 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3899..3918) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4018..4040 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4078..4096) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4164..4268 /gene="bla" /label=AmpR promoter CDS 4269..5129 /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(4487..4506) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5300..5888 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5789..5808 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgtgtc tagactgcag agggccctgc 181 gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg 241 accgaccccg acccactgga caagcaccca acccccattc cccaaattgc gcatccccta 301 tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca 361 ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa 421 ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc 481 gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac 541 gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc 601 agcggaggag tcgtgtcgtg cctgagagcg cagtcgagaa ggtaccggat cctctagagt 661 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg 721 attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac 781 gaagttatgg cgcgccttta aacgcattag tcttccaatt gaaaaaagtg atttaattta 841 taccatttta attcagcttt gtaaaaatgt atcaaagaga tagcaaggta ttcagtttta 901 gtaaacaaga taattgctcc taaagtagcc ccttgaattc cgaggcagta ggcaaacaca 961 gtattctgta ccatactaca tctgtggctt cactagtatg gtacagaata ctgtgtgcgc 1021 tcactgtcaa cagcaatata ccttctcgag ccttctgttg ggttaacctg aagaagtaat 1081 cccagcaagt gtttccaaga tgtgcaggca acgattctgt aaagtactga agcctcattc 1141 aaacatagta tatgtgctgc cgaagcgagc acttaacaag gcttgcggcc gctacttgta 1201 cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg 1261 atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc 1321 gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg 1381 cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg 1441 cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag 1501 gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc 1561 gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt 1621 gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg 1681 gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca 1741 gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc 1801 atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc 1861 gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca tggtggcgac 1921 cggtagcgct agcataactt cgtataaagt atcctatacg aagttatttg ccttaaccca 1981 gaaattatca ctgttattct ttagaatggt gcaaagaata acttcgtata atgtatgcta 2041 tacgaagtta tgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt 2101 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg 2161 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt 2221 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg 2281 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc 2341 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg 2401 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt 2461 tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc 2521 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg 2581 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga 2641 tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg 2701 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc 2761 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta 2821 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg 2881 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac 2941 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg 3001 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt 3061 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc 3121 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt 3181 gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact 3241 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg 3301 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg 3361 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac 3421 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 3481 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 3541 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 3601 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta 3661 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 3721 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg 3781 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 3841 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta 3901 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 3961 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 4021 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc 4081 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag 4141 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 4201 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 4261 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 4321 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 4381 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 4441 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 4501 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 4561 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 4621 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 4681 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 4741 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 4801 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 4861 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 4921 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 4981 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 5041 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 5101 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 5161 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 5221 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 5281 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 5341 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 5401 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 5461 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 5521 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 5581 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 5641 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 5701 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 5761 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 5821 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 5881 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 5941 ctcacatgt //