LOCUS Exported 4111 bp ds-DNA linear SYN 17-MAY-2021 DEFINITION Donor for precise CRISPR directed genomic integration using the GeneWeld method. ACCESSION . VERSION . KEYWORDS pPRISM-TagRFP-gcry-mRFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4111) AUTHORS Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Weiss TJ, Torrie ME, Vollbrecht MK, Lan M, McKeighan KC, Ming Z, Wehmeier A, Mikelson CS, Haltom JA, Kwan KM, Chien CB, Balciunas D, Ekker SC, Clark KJ, Webber BR, Moriarity B, Solin SL, Carlson DF, Dobbs DL, McGrail M, Essner JJ. TITLE GeneWeld: a method for efficient targeted integration directed by short homology JOURNAL bioRxiv 431627 REFERENCE 2 (bases 1 to 4111) AUTHORS . TITLE Direct Submission JOURNAL Exported May 17, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4111 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 10..66 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 73..783 /codon_start=1 /product="monomeric derivative of red fluorescent protein from Entacmaea quadricolor (Merzlyak et al., 2007)" /label=TagRFP /note="mammalian codon-optimized" /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDM ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA RYCDLPSKLGHKLN" CDS 2657..2677 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2681..3358 /codon_start=1 /product="monomeric derivative of DsRed (Campbell et al., 2002)" /label=mRFP1 /note="mammalian codon-optimized" /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS TGA" primer_bind complement(2811..2829) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 3079..3098 /label=mCherry-F /note="mCherry, forward primer" primer_bind 3273..3296 /label=DsRed1-C /note="DsRed1, forward primer" polyA_signal 3368..3592 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" polyA_signal 3662..3743 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 3867..3948 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" ORIGIN 1 cttcccggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga agaaaacccc 61 ggtcctctcg aggtgtctaa gggcgaggaa ctgattaagg agaacatgca catgaagctg 121 tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag 181 ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc 241 gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat caaccacacc 301 cagggcatcc ccgacttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc 361 accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc 421 tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg 481 cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc tgacggcggc 541 ctggaaggca gaagcgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac 601 ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac 661 tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag 721 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca caaacttaat 781 tgaaactagt aattaagtct cagccaccgt taactgaaca tgtcaaaacc tgtggagact 841 gttgagattt gatgttctga aaagataaag cctataaata aaatgttgcc caaatttcct 901 gcctgatgtt tttctttgtc tttgctacat ggctttgctg ctcggatcgg ctcactctgt 961 gtatgccacg ttcactttgt actctccttc tcacggtagg tttattattt ttagatgtgc 1021 agttagtttc tgtgaaataa cacaccacac actgatattg tctgtgcatt gacttggtga 1081 gtgcacattg tttttgatct tgacatattt atatttgatt gatcaggtga actgtgtgaa 1141 tctaaagtgc tccatacaga tgttctgcat tgaaaatatt ctcattttat tagtggaagt 1201 gagtgtatgc cacatccaat caatttcagc aaacacccca gtatgattta atgcaaaaaa 1261 atgaaggtat caaacacgca ttactacttt gcagttaaat atttaacatt tattccaaca 1321 cgaaaaaaag cagtaaataa cactttgaca aacacgtcag gacatcttat ttttgtcacc 1381 ctcacaggca atttagtata atatattata tatatatata tatcatataa taatattcag 1441 tataatatat atatatatat catattataa tattcagtat aatataaaac acaaacacat 1501 atatgtataa tataatataa catttttatt tattgagatg cctctatgga ccgtgttata 1561 agaagtaaag atcaggagaa gtaaacatga agtgtaatta tgaatactga tgttaaatta 1621 agctatgatg agttttcact gttaatttac catctcaatt aaatgttgat gcctccatga 1681 ccaagttaag cagatgagac tgagacaact gtagaagaca agatgttcac tttgctgaat 1741 atagctggct tgacagttat ctatgactct ataaatatat atatattttt ttttttataa 1801 aatgatttat ttataactat atatccattt ctcagacagg tgcttcatat ccctcactcc 1861 cgtagctgtc catgctggat ctgtccccgt tgtttttaaa aagctaaata agttattaac 1921 atgactgcat ccagcgagcc aaacctgtct ggtgtacagc taccagagaa gcttgagatc 1981 ctagtcaccg gcgttcctag ttctttaaac tgttacaaag tgtttggacc agcaactgaa 2041 ttgcactgta aattaccaca tcagggtctc cggggtcaac gctgctaagc aatggactgg 2101 aaaatttgtg acattgagtt tttggaataa ctgggaatgc tggataactc tttgggtaaa 2161 gctatggaac aaaagtacca gcatgaacta agaaatgact gtcacagcac aacatcaaac 2221 acaatgcaag acaaaggttc tctgcttttg ccttacttga cagaaaaagt aagctctagg 2281 ttcatcgcat gcagacagca gcaatacatg acatatataa aggctggctc ttttctccct 2341 tccattgaac tgaaacttcc actcagtcag acttgcgggg gatccacagc ctgactaact 2401 cgacaaaccc ccccaaacct aaggtgagtt gatctttaag ctttttacat tttcagctcg 2461 catatatcaa ttcgcgcgca ttgtcacact agcacccact agcggtcaga ctgcagattg 2521 cagcacgaaa caggaagctg actccacatg gtcacatgct cactgaagtg ttgacttccc 2581 tgacagctgt gcactttcta aaccggtttt ctcattcatt tacagttcag ccggaattca 2641 cctcgagacc atggctccaa agaagaagcg taaggtaacc atggcctcct ccgaggacgt 2701 catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg gccacgagtt 2761 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa 2821 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc agttccagta 2881 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt 2941 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt 3001 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac 3061 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctccac 3121 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga ggctgaagct 3181 gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca agaagcccgt 3241 gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc acaacgagga 3301 ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg gcgcctaaag 3361 ctgagatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 3421 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 3481 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 3541 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 3601 gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc acaactagaa 3661 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca 3721 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc 3781 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtcattgat 3841 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 3901 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat 3961 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa 4021 aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag cgatgcatgg 4081 gagctctgac gtacgttgtc tagcaaggaa g //