LOCUS Exported 7461 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Cre-inducible shRNA. ACCESSION . VERSION . KEYWORDS pAAV-Sico-Red SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7461) AUTHORS Jung JY, Lee SE, Hwang EM, Lee CJ TITLE Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System. JOURNAL Exp Neurobiol. 2016 Jun;25(3):120-9. doi: 10.5607/en.2016.25.3.120. Epub 2016 Jun 22. PUBMED 27358580 REFERENCE 2 (bases 1 to 7461) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7461 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 71..211 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 71..200 /label=AAV2 ITR rep_origin 286..741 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(373..392) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 583..604 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(758..777) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 877..899 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(937..955) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1023..1127 /gene="bla" /label=AmpR promoter CDS 1128..1988 /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(1346..1365) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2159..2747 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2648..2667 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 2809..2938 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 2953..4131 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 3184..4122 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 4079..4099 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 4157..4867 /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(4302..4320) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 4570..4589 /label=mCherry-F /note="mCherry, forward primer" polyA_signal 4989..5110 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5026..5045) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5080..5099 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 5378..5398 /label=mU6-F /note="Mouse U6 promoter, forward primer" protein_bind 5416..5449 /label=loxP511 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GTATACAT). loxP511 sites are compatible with each other, but incompatible with wild-type loxP sites." enhancer 5549..5852 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5853..6056 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6007..6027 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 6059..6079 /gene="mutant version of attB" /label=attB5 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" regulatory 6087..6096 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 6093..6809 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(6138..6159) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(6399..6418) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 6746..6767 /label=EGFP-C /note="EGFP, forward primer" protein_bind 6820..6853 /label=loxP511 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GTATACAT). loxP511 sites are compatible with each other, but incompatible with wild-type loxP sites." primer_bind 6921..6937 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 6922..6938 /label=KS primer /note="common sequencing primer, one of multiple similar variants" protein_bind complement(6983..7007) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" polyA_signal 7016..31 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(7215..7234) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" ORIGIN 1 ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc 61 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 121 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 181 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 241 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 301 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 361 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 421 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 481 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 541 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 601 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga 661 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 721 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 781 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 841 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 901 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 961 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 1021 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 1081 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 1141 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 1201 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 1261 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 1321 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 1381 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 1441 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 1501 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 1561 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 1621 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 1681 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 1741 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 1801 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 1861 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 1921 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 1981 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 2041 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 2101 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 2161 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 2221 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 2281 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 2341 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 2401 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 2461 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 2521 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 2581 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 2641 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 2701 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 2761 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc 2821 tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc 2881 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc 2941 ggccgcacgc gtggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 3001 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta 3061 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg 3121 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca 3181 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc 3241 gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt 3301 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga 3361 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc 3421 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg 3481 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg 3541 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg 3601 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct 3661 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc 3721 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc 3781 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa 3841 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc 3901 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg 3961 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg 4021 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc 4081 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg attcgaacat 4141 cgattgaatt cccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga 4201 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga 4261 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa 4321 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa 4381 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg 4441 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga 4501 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc 4561 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat 4621 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg 4681 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc 4741 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat 4801 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta 4861 caagtaagcg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac 4921 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg 4981 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 5041 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 5101 atgtatctta aggcgtacaa gttgtacaaa aaagcaggct tctagagatc cgacgcgcca 5161 tctctaggcc cgcgccggcc ccctcgcaca gacttgtggg agaagctcgg ctactcccct 5221 gccccggtta atttgcatat aatatttcct agtaactata gaggcttaat gtgcgataaa 5281 agacagataa tctgttcttt ttaatactag ctacatttta catgataggc ttggatttct 5341 ataagagata caaatactaa attattattt taaaaaacag cacaaaagga aactcaccct 5401 aactgtaaag taattataac ttcgtatagt ataaattata cgaagttata agccttggtt 5461 ttttgaattc cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat 5521 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 5581 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 5641 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 5701 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 5761 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 5821 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 5881 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 5941 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc 6001 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggca 6061 actttgtata caaaagttga ccggtcgcca ccatggtgag caagggcgag gagctgttca 6121 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 6181 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 6241 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 6301 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 6361 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 6421 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 6481 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 6541 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 6601 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 6661 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca 6721 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 6781 tcactctcgg catggacgag ctgtacaagt agcggccgca taacttcgta tagtataaat 6841 tatacgaagt tataagcctt gttttttgcc aacttgtcaa tgaattcaag agattcattg 6901 acaagttggc aaattttttc tcgaggtcga cggtatcgat aagctcgctt cacgagatca 6961 tgtttaaggg ttcccgttcc aaacccagct ttcttgtaca aagtggtaga tctacgggtg 7021 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc 7081 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta 7141 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg 7201 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac 7261 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg 7321 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt 7381 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc 7441 tcccaaattg ctgggattac a //