LOCUS Exported 5753 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes a HR repair template for knock-in a YFP expression cassette downstream of the poly(A) signal of human NEAT1_1 isoform. Best used with px330-NEAT1m_v1 vector.. ACCESSION . VERSION . KEYWORDS pYFP-NEAT1m_v1-RT SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5753) 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 5753) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5753 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 548..851 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 852..1055 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1006..1026 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 1094..1103 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1100..1816 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1145..1166) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1406..1425) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1753..1774 /label=EGFP-C /note="EGFP, forward primer" misc_feature 1817..1882 /label=MCS /note="multiple cloning site" polyA_signal 2006..2127 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2043..2062) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2097..2116 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2669..3124) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2806..2827) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3018..3037 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3151..3255 /gene="bla" /label=AmpR promoter promoter 3257..3614 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3280..3300) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3465..3600 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3527..3546 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3649..4443 /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(3703..3722) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4313..4332 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4631..4650) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4675..4722 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5051..5639 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5540..5559 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tagttatgtt ctgagctgcg tctattgaat tggtaaagta ataccaatgg ctttttatca 61 tttccttctt ccctttaagt ttcacttgaa attttaaaaa tcatggttat ttttatcgtt 121 gggatctttc tgtcttctgg gttccatttt ttaaatgttt aaaaatatgt tgacatggta 181 gttcagttct taaccaatga cttggggatg atgcaaacaa ttactgtcgt tgggatttag 241 agtgtattag tcacgcatgt atggggaagt agtctcgggt atgctgttgt gaaattgaaa 301 ctgtaaaagt agatggttga aagtactggt atgttgctct gtatggtaag aactaattct 361 gttacgtcat gtacataatt actaatcact tttcttcccc tttacagcac aaataaagtt 421 tgagttctaa actcattaga attgttgtat tgctatgtta catttctcga cccctatcac 481 attgccttca taacgaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 541 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 601 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 661 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 721 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 781 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 841 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 901 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 961 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 1021 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 1081 agcgctaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc 1141 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg 1201 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct 1261 gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg 1321 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt 1381 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa 1441 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga 1501 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat 1561 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga 1621 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt 1681 gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga 1741 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat 1801 ggacgagctg tacaagtccg gactcagatc tcgagctcaa gcttcgaatt ctgcagtcga 1861 cggtaccgcg ggcccgggat ccaccggatc tagataactg atcataatca gccataccac 1921 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca 1981 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata 2041 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 2101 tttgtccaaa ctcatcaatg tatcttaact ttggatgtat cttcatattg tagatttagg 2161 tctagatttg ctagctccaa gtaattaagg ccatgtagga gagcatggta accacagata 2221 gaactggtat tatcccaagt ggtctgcaga ctgctgagtg gggatgggat ctgctctctg 2281 ttgagagttg gtaatcattg gtttgaaatg tgatgaaacc actcaagcca atgaaggtgg 2341 gtgtgtaggt ggggagtact ttgccataat attttaaaac attacctggt tagagttcta 2401 agtggtactt atttttgttt ggttagggga aagcctgaat aaaaacagaa atggacacat 2461 aatatgcata ttccatagtc tttgggaggc tggaatgtgc ctgggatttg ggtctaagtg 2521 tatgcgtaat tcttacctca ctaaagaatt tgccttgttt ttttcctttt ggtgagtgac 2581 taaaacgtct gggcttccct gtgtgcgtgc tacagtaagc aagcagaggc tgtgcaaagg 2641 tgtgagcagg atcacgtgga atctgggtaa attgtaagcg ttaatatttt gttaaaattc 2701 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc 2761 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag 2821 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc 2881 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa 2941 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg 3001 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt 3061 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc 3121 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 3181 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 3241 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt 3301 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 3361 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 3421 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc 3481 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg 3541 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc 3601 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa 3661 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg 3721 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc 3781 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca 3841 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc 3901 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca 3961 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat 4021 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca 4081 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg 4141 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc 4201 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct 4261 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct 4321 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac 4381 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc 4441 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag 4501 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg 4561 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg 4621 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa 4681 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt 4741 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg 4801 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca 4861 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt 4921 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 4981 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 5041 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 5101 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 5161 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 5221 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 5281 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 5341 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 5401 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 5461 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 5521 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 5581 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 5641 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 5701 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cat //