LOCUS Exported 5797 bp ds-DNA circular SYN 20-MAY-2022 DEFINITION Repair template for the N-terminal tagging of RhoA with mNeonGreen in human cells using CRISPR/Cas9.. ACCESSION . VERSION . KEYWORDS pODN-mNG-RHOA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5797) AUTHORS Husser MC, Ozugergin I, Resta T, Martin VJJ, Piekny A TITLE Imaging tools generated by CRISPR/Cas9 tagging reveal cytokinetic diversity in mammalian cells JOURNAL bioRxiv (2022) REFERENCE 2 (bases 1 to 5797) AUTHORS . TITLE Direct Submission JOURNAL Exported May 20, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5797 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(171..1031) /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 794..813 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1032..1136) /gene="bla" /label=AmpR promoter primer_bind 1204..1222 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind 1528..1547 /label=T7 /note="T7 promoter, forward primer" promoter 1528..1546 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 2692..3399 /codon_start=1 /product="bright monomeric yellow-green fluorescent protein derived from LanYFP (Shaner et al., 2013)" /label=mNeonGreen /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ KAFTDVMGMDELYK" regulatory 2947..2956 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(4852..4882) /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind 4897..4918 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5038..5055) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5209..5797) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5289..5308) /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 1141 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 1201 aaaaataggc gtatcacgag gccgcccctg cagccgaatt atattatttt tgccaaataa 1261 tttttaacaa aagctctgaa gtcttcttca tttaaattct tagatgatac ttcatctgga 1321 aaattgtccc aattagtagc atcacgctgt gagtaagttc taaaccattt ttttattgtt 1381 gtattatctc taatcttact actcgatgag ttttcggtat tatctctatt tttaacttgg 1441 agcaggttcc attcattgtt tttttcatca tagtgaataa aatcaactgc tttaacactt 1501 gtgcctgaac accatatcca tccggcgtaa tacgactcac tatagggaga gcggccgcca 1561 gatcttccgg atggctcgag tttttcagca agattaccct gcttgacaaa ttgtttccnt 1621 tttttttttt ttttttgtga gatggagtct tgctctgtcc cccaggctgg tgtgcagtgg 1681 cccgatcttg gctcactgca agctctgtct cctgggttca cgccattctc ctgcctcagc 1741 ctcccaagta gctgggacta caggtgcctg ccaccatgcc cggctagtta tttgtatttt 1801 tagtagagat ggggtttctc cgtgttagcc aggatggtct cgatctcctg acctcgtgat 1861 ccacccgcct cggcctccca aagtgctggg attataggcg tgagccactg cgcctggctc 1921 aattgatttt ttaatgttaa tattgtatcc tgcaacctta ctgaacttgt ttattctaaa 1981 agttttcttt aggatttccc acattacaaa atcatctcat gtgtgaattg agatagtttt 2041 acttcttcct ttccaatttg gatgtctttt atttcatttt cttaccttat tgccctggct 2101 aggacctcca gtatcctgct ggatagaagt agcaagaaca ggccaggcgt ggtggctcac 2161 gcctgtaatc ccagcacttt gggaggctga ggcaggtgga tcacctgagg tcaggagttc 2221 aagaccagcc tggccaacaa ggtgaaaccc catctctatc aaaaattagc tggatgtggt 2281 ggtacgtgcc tgtaatccca gctacttggg agtctgaggc aggagaattg cttgaacccg 2341 ggaggctgga ggttgcagtg aactgagatt gggccagtgc actccagcct gggcgacaga 2401 gtgagactcc gtctcaaaaa aaaaaagaag tggcaagaac agacattctt gtcttgttcc 2461 tgatcttagg gggcagacct ttttttcagt cttttaccat taagtataat gttagttacg 2521 agtttttaat gtataacctt ttggtgccag gtggatcggc gtactagaag tttgaatttg 2581 agtatgctaa tagaggttta tgccccatgg ttaccaaagc atgtgtcatc ctgtaagcta 2641 cctatgactt cttgtgcatt gcaggtaata tctgtgtttt gtgtttcagc aatggtgagc 2701 aagggcgagg aggataacat ggcctctctc ccagcgacac atgagttaca catctttggc 2761 tccatcaacg gtgtggactt tgacatggtg ggtcagggca ccggcaatcc aaatgatggt 2821 tatgaggagt taaacctgaa gtccaccaag ggtgacctcc agttctcccc ctggattctg 2881 gtccctcata tcgggtatgg cttccatcag tacctgccct accctgacgg gatgtcgcct 2941 ttccaggccg ccatggtaga tggctccgga taccaagtcc atcgcacaat gcagtttgaa 3001 gatggtgcct cccttactgt taactaccgc tacacctacg agggaagcca catcaaagga 3061 gaggcccagg tgaaggggac tggtttccct gctgacggtc ctgtgatgac caactcgctg 3121 accgctgcgg actggtgcag gtcgaagaag acttacccca acgacaaaac catcatcagt 3181 acctttaagt ggagttacac cactggaaat ggcaagcgct accggagcac tgcgcggacc 3241 acctacacct ttgccaagcc aatggcggct aactatctga agaaccagcc gatgtacgtg 3301 ttccgtaaga cggagctcaa gcactccaag accgagctca acttcaagga gtggcaaaag 3361 gcctttaccg atgtgatggg catggacgag ctgtacaagg gaggttcagg aggctccatg 3421 gcagctattc gcaaaaaact ggtgattgtt ggtgatggag cctgtggaaa gacatgcttg 3481 ctcatagtct tcagcaagga ccagttccca gaggtgtatg tgcccacagt gtttgagaac 3541 tatgtggcag atatcgaggt ggatggaaag caggtgagta tacttttcat aacaactgat 3601 gccattttcc atgttagaat atttatggag catgtagaga attagagctt tttgcctctt 3661 cagtatacca tataacaaaa atgttggaaa gaagaatcca ttctcatcct gccatcttag 3721 tccaagtttg ttcttttttt ttttttggag acggagtctc attctgttgc ccaggctaga 3781 gtgcagtggc tcggtcttgg ctcactgcaa cctctgcctc ccaggttcaa gtgattctcc 3841 tgcctcagcc tcccaaatag cagggattac aggcatgttg ccaccatgct gagctcattt 3901 ttcgtttagt agagatgggg tttcaccagg ctggtctcaa acttctgacc tcgggtgatc 3961 cacccacctc agcctcccaa agtgctggga ttacaggagt gagccactgt gcccagcctt 4021 cccccaggtt taaaggattc tcctgcctca gcctccccag tagctgggat tacgggaata 4081 tgccaccaca cctggctgat ttttgtattt ttagtagaaa tggggtttca ctacgttggc 4141 caggctggtc atgaactcct gaccgcaggt gattggctgt tttttcattt ttaactgcta 4201 ctcatccaga ctttgtttac attcatatgt atttgacatg gtgtccttag catatgtgtt 4261 ttttttttgt tttgttttgt tgtttgagac agagtctcgc tctgtcgccc aggctggagt 4321 gcagtggtac aatctcagct cgctgcaacc tccacctccc gggttcaagc aattcttttg 4381 cctcagcctc cagagtagag tagctgggat tacaggcatc tttctagaag atctcctaca 4441 atattctcag ctgccatgga aaatcgatgt tcttctttta ttctctcaag attttcaggc 4501 tgtatattaa aacttatatt aagaactatg ctaaccacct catcaggaac cgttgtaggt 4561 ggcgtgggtt ttcttggcaa tcgactctca tgaaaactac gagctaaata ttcaatatgt 4621 tcctcttgac caactttatt ctgcattttt tttgaacgag gtttagagca agcttcagga 4681 aactgagaca ggaattttat taaaaattta aattttgaag aaagttcagg gttaatagca 4741 tccatttttt gctttgcaag ttcctcagca ttcttaacaa aagacgtctc ttttgacatg 4801 tttaaagttt aaacctcctg tgtgaaatta ttatccgctc ataattccac acattatacg 4861 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 4921 tgcgttgcgc tcactgccaa ttgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 4981 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 5041 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 5101 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 5161 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 5221 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 5281 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 5341 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 5401 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 5461 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 5521 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 5581 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 5641 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 5701 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 5761 caagcagcag attacgcgca gaaaaaaagg atctcaa //