LOCUS Exported 5414 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Homology arms and linker-mEGFP sequence for N-terminus tagging of human LMNB1. ACCESSION . VERSION . KEYWORDS AICSDP-10:LMNB1-mEGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5414) AUTHORS Roberts B, Haupt A, Tucker A, Grancharova T, Arakaki J, Fuqua MA, Nelson A, Hookway C, Ludmann SA, Mueller IA, Yang R, Horwitz R, Rafelski SM, Gunawardane RN TITLE Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. JOURNAL Mol Biol Cell. 2017 Oct 15;28(21):2854-2874. doi: 10.1091/mbc.E17-03-0209. Epub 2017 Aug 16. PUBMED 28814507 REFERENCE 2 (bases 1 to 5414) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5414 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 364..386 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 378..395 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 379..395 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 1415..2131 /codon_start=1 /product="enhanced GFP with monomerizing A206K mutation (Zacharias et al., 2002)" /label=mEGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1460..1481) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1721..1740) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2068..2089 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(3193..3209) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3193..3209) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3206..3228) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3217..3233 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(3241..3271) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3286..3307 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3424..3441) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3595..4183) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3675..3694) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4354..5214) /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 4977..4996 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5215..5319) /gene="bla" /label=AmpR promoter primer_bind 5387..5405 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggattaaag 421 gctggtactt ggaacctgca agccgtgcat ttggaacctc ggactcaagt gcctattacg 481 taattccaca gcgtcccggc ctccaggccg tttcccgagc cctccagcgg agcgggggat 541 aaggttacca cgcccgcggt ggccggggac actctgagtt tcgcgtgtgg cttttaggga 601 cgtttatatt tgaatttccc tgaaccgccg agtgtgggcg gtggcgcaga tccgtcccgg 661 aaacctccgg gctccttccc gcctttctca ggcccggccc ctccaagggg tccccgcggg 721 gcggcgggag ggccctgggc ccagagccgc gcgggtgggc agtcccaggc gtccttcctt 781 acagccctga gcctggtccg ggaaccgccc agccgggagg gccgagctga cggttgccca 841 agggccagat tttaaattta caggcccggc ccccgaaccg ccgaagcgcg ctgcctgctc 901 cccattggcc catggtagtc acgtggaggc gccggggcgt gccggccatg ttggggagtg 961 cggcgccgcg gcccgcgcca cctccgcccc ccgcggcttg cctccagccc gcccctcccg 1021 gccctcctcc ccccgcccgc cgctccgtgc agcctgagag gaaacaaagt gctgcgagca 1081 ggagacggcg gcggcgcgaa ccctgctggg cctccagtca ccctcgtctt gcattttccc 1141 gcgtgcgtgt gtgagtgggt gtgtgtgttt tcttacaaag ggtatttcgc gatcgatcga 1201 ttgattcgta gttccccccc gcgcgccttt gccctttgtg ctgtaatcga gctcccgcca 1261 tcccaggtgc ttctccgttc ctctaaacgc cagcgtctgg acgtgagcgc aggtcgccgg 1321 tttgtgcctt cggtccccgc ttcgccccct gccgtcccct ccttatcacg gtcccgctcg 1381 cggcctcgcc gccccgctgt ctccgccgcc cgccatggtg agcaagggcg aggagctgtt 1441 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 1501 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 1561 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 1621 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 1681 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 1741 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 1801 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 1861 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 1921 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 1981 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag 2041 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 2101 gatcactctc ggcatggacg agctgtacaa gagcggcctg agaagcagag cccaggccag 2161 cgcgactgcg acccccgtgc cgccgcggat gggcagccgc gctggcggcc ccaccacgcc 2221 gctgagcccc acgcgcctgt cgcggctcca ggagaaggag gagctgcgcg agctcaatga 2281 ccggctggcg gtgtacatcg acaaggtgcg cagcctggag acggagaaca gcgcgctgca 2341 gctgcaggtg acggagcgcg aggaggtgcg cggccgtgag ctcaccggcc tcaaggcgct 2401 ctacgagacc gagctggccg acgcgcgacg cgcgctcgac gacacggccc gcgagcgcgc 2461 caagctgcag atcgagctgg gcaagtgcaa ggcggaacac gaccagctgc tcctcaagtg 2521 agtgctagct ggcggccgcg ttagcgccaa ggaggggcgg gggcgcaacc gcggcgacca 2581 gctcaccggg ttctgccgtg gggagggagc agaggccagg atgcacgcgt ccttctgaag 2641 gaacagggtc tcggtctccg gaaaggagaa agaatctaga gttcatagcg gagcaggggt 2701 cgcggagggg gctcgagctg tagcgctggg gggccgtgat gcccatttct agattttgga 2761 tacccgctgg gacgtggtaa gtgcgcgcct gggactgccg agaaggagct cccgctttcg 2821 cactcgaatc cggggagccg gcgcggagag gcggcccctc aggccccagg tgcggggagc 2881 tggagcgcga gcgcgcgctc gcgtgcgcgc cccagtttcc ggccggcgcg agacaaagcg 2941 tctagcggat ttgcagtgcc gggatgggcg gccggggagg actggcagcc cgcctctaga 3001 atgaatgagc ttcgcgcggg cagagagagg aaggggaggg accttcccgc agcatccgcg 3061 tctcctgggg gtgggtcccg ctttggcgcg ctcagtcttg gccctgtgac gttttgcgaa 3121 gattctacgc ctgctttagg cgggagagag aggcggagat cccaatggcg cgccgagctt 3181 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 3241 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 3301 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 3361 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 3421 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 3481 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 3541 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 3601 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 3661 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 3721 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 3781 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 3841 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 3901 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 3961 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4021 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4081 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4141 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4201 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 4261 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 4321 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 4381 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 4441 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 4501 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 4561 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 4621 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 4681 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 4741 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 4801 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 4861 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 4921 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 4981 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 5041 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 5101 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 5161 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 5221 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 5281 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 5341 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 5401 gaggcccttt cgtc //