LOCUS Exported 5906 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes a HR repair template for knock-in a YFP expression cassette at the promoter of human NEAT1 gene. Best used with px330-NEAT1pr_v2 vector, but also works with px330-NEAT1pr_v1.. ACCESSION . VERSION . KEYWORDS pYFP-NEAT1pr_v2-RT SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5906) AUTHORS Li R, Harvey AR, Hodgetts SI, Fox AH TITLE Functional dissection of NEAT1 using genome editing reveals substantial localisation of the NEAT1_1 isoform outside paraspeckles. JOURNAL RNA. 2017 Mar 21. pii: rna.059477.116. doi: 10.1261/rna.059477.116. PUBMED 28325845 REFERENCE 2 (bases 1 to 5906) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5906 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 703..1006 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1007..1210 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1161..1181 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 1249..1258 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1255..1971 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1300..1321) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1561..1580) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1908..1929 /label=EGFP-C /note="EGFP, forward primer" misc_feature 1972..2037 /label=MCS /note="multiple cloning site" polyA_signal 2161..2282 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2198..2217) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2252..2271 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2822..3277) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2959..2980) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3171..3190 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3304..3408 /gene="bla" /label=AmpR promoter promoter 3410..3767 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3433..3453) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3618..3753 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3680..3699 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3802..4596 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3856..3875) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4466..4485 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4784..4803) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4828..4875 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5204..5792 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5693..5712 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tagttatgga cgctatcagc ccgcagcagg gtttcctggc cagagaaacc gcctgttggg 61 gtgcggagcg ttccacgtcc cctcgcagca ccccgctggc cttctgggcc gccccgcccc 121 caacccgcgc ccgggcgctg acgtcatcgg ccgagcccga ctcggaacca ccgcccgaaa 181 gtcacgcgcg cctcccggcg ggacgcgccc tctgcagaga tccctccgcc gccgcctgga 241 attttccaga tgtcctgccg gcccttcttt cgggcctcgg ctgggcgtgg tgacctcaag 301 cgaccccggc ggctcccgtc gcccactcaa gagggcgccc ccgcccgcca cgcccccagc 361 ccctctcggg gaagcgcgtc ccccgcccga cctcaacaac atccgggaag aaaaggggtc 421 ttcttcctca tggcattcgc ctcccaaatg tcaccttgag cgctgtccgc gatcccaaaa 481 agcactgtta aagagaagcg gggatacact ggggtccttg cgtgggggcc gcctgggaga 541 ccatgcaccg cccgggagtc tctccgggca gggtcgggga gggacttttt tgccggtggc 601 cgtggaggaa tcgtcccgtt gagcaatgac cccggtgacg cggctgaggg catagtaatc 661 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 721 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 781 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 841 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 901 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 961 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 1021 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 1081 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 1141 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 1201 aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc caccatggtg 1261 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 1321 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 1381 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 1441 accaccttcg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac 1501 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag 1561 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 1621 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 1681 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc 1741 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac 1801 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 1861 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 1921 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggactc 1981 agatctcgag ctcaagcttc gaattctgca gtcgacggta ccgcgggccc gggatccacc 2041 ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta 2101 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 2161 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 2221 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 2281 taacgccagg gagggatgcg cgcctgggtg tagttgtggg ggaggaagtg gctagctcag 2341 ggcttcaggg gacagacagg gagagatgac tgagttagat gagactaggg ggcgggctgg 2401 gggtgcgaga aggaagcttg gcaaggagac taggtctagg gggaccacag tggggcaggc 2461 tgcatggaaa atatccgcag ggtcccccag gcagaacagc cacgctccag gccaggctgt 2521 ccctactgcc tggtggaggg ggaacttgac ctctgggagg gcgccgctct tgcatagctg 2581 agcgagcccg ggtgcgctgg tctgtgtgga aggaggaagg cagggagagg tagaaggggt 2641 ggaggagtca ggaggaatag gccgcagcag ccctggaaat gatcaggaag gcaggcagtg 2701 ggtgcagggc tgcaggaggg ccgggagggc taatcttcaa cttgtccatg ccagcagccc 2761 ctttttttcc agaccaaggg ctgtgaaccc gcctggggat gagcctggtc ttgtggaact 2821 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca 2881 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag 2941 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc 3001 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc 3061 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc 3121 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa 3181 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc 3241 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat 3301 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 3361 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa 3421 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag 3481 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag 3541 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc 3601 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc 3661 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat 3721 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag 3781 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc 3841 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 3901 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 3961 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg 4021 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 4081 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 4141 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 4201 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 4261 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 4321 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg 4381 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 4441 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 4501 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 4561 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga 4621 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg 4681 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg 4741 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga 4801 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg 4861 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc 4921 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc 4981 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag 5041 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 5101 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 5161 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 5221 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 5281 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 5341 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 5401 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 5461 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 5521 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 5581 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 5641 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 5701 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 5761 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 5821 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 5881 tggataaccg tattaccgcc atgcat //