LOCUS Exported 6967 bp ds-DNA circular SYN 12-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 6967) 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 6967) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6967 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 275..296 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 311..341 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 349..365 /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)." primer_bind 354..376 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 373..389 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 373..389 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 385..750 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS VKASRRRGRKPRSSRGV" primer_bind 408..428 /label=T3 /note="T3 promoter, forward primer" promoter 410..428 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter complement(476..494) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(504..521) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(504..520) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(513..535) /label=M13/pUC Forward /note="In lacZ gene" CDS 743..1453 /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 3327..3347 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3351..4028 /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(3481..3499) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 3749..3768 /label=mCherry-F /note="mCherry, forward primer" primer_bind 3943..3966 /label=DsRed1-C /note="DsRed1, forward primer" polyA_signal 4038..4262 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" polyA_signal 4332..4413 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 4537..4618 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter 4935..4953 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5229..5248) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 5240..6049 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(5307..5326) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 6192..6780 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6681..6700 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6934..6951 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 aaatggtacc gagcagaggt gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa 61 gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa tcttacttga 121 gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag taaaagtaaa 181 agcaagaaag aaaactagag tggtctccct ttagtgaggg tcaattgatg ggaggcgttc 241 gggccacagc ggacacgcag gtgcgcaacg caattaatgt gagttagctc actcattagg 301 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 361 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc 421 actaaaggga acaaaagctg gaggacctcg agggggggcc cggtacccaa ttcgccctat 481 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac 541 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 601 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 661 acctgccaca cttcccggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga 721 agaaaacccc ggtcctctcg aggtgtctaa gggcgaggaa ctgattaagg agaacatgca 781 catgaagctg tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg 841 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc 901 tctccccttc gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat 961 caaccacacc cagggcatcc ccgacttctt taagcagtcc ttccctgagg gcttcacatg 1021 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct 1081 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg 1141 ccctgtgatg cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc 1201 tgacggcggc ctggaaggca gaagcgacat ggccctgaag ctcgtgggcg ggggccacct 1261 gatctgcaac ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc 1321 cggcgtctac tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta 1381 cgtcgagcag cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca 1441 caaacttaat tgaaactagt aattaagtct cagccaccgt taactgaaca tgtcaaaacc 1501 tgtggagact gttgagattt gatgttctga aaagataaag cctataaata aaatgttgcc 1561 caaatttcct gcctgatgtt tttctttgtc tttgctacat ggctttgctg ctcggatcgg 1621 ctcactctgt gtatgccacg ttcactttgt actctccttc tcacggtagg tttattattt 1681 ttagatgtgc agttagtttc tgtgaaataa cacaccacac actgatattg tctgtgcatt 1741 gacttggtga gtgcacattg tttttgatct tgacatattt atatttgatt gatcaggtga 1801 actgtgtgaa tctaaagtgc tccatacaga tgttctgcat tgaaaatatt ctcattttat 1861 tagtggaagt gagtgtatgc cacatccaat caatttcagc aaacacccca gtatgattta 1921 atgcaaaaaa atgaaggtat caaacacgca ttactacttt gcagttaaat atttaacatt 1981 tattccaaca cgaaaaaaag cagtaaataa cactttgaca aacacgtcag gacatcttat 2041 ttttgtcacc ctcacaggca atttagtata atatattata tatatatata tatcatataa 2101 taatattcag tataatatat atatatatat catattataa tattcagtat aatataaaac 2161 acaaacacat atatgtataa tataatataa catttttatt tattgagatg cctctatgga 2221 ccgtgttata agaagtaaag atcaggagaa gtaaacatga agtgtaatta tgaatactga 2281 tgttaaatta agctatgatg agttttcact gttaatttac catctcaatt aaatgttgat 2341 gcctccatga ccaagttaag cagatgagac tgaggacaac tgtagaagac aagatgttca 2401 ctttgctgaa tatagctggc ttgacagtta tctatgactc tataaatata tatatatttt 2461 ttttttataa aatgatttat ttataactat atatccattt ctcagacagg tgcttcatat 2521 ccctcactcc cgtagctgtc catgctggat ctgtccccgt tgtttttaaa aagctaaata 2581 agttattaac atgactgcat ccagcgagcc aaacctgtct ggtgtacagc taccagagaa 2641 gcttgagatc ctagtcaccg gcgttcctag ttctttaaac tgttacaaag tgtttggacc 2701 agcaactgaa ttgcactgta aattaccaca tcagggtctc cggggtcaac gctgctaagc 2761 aatggactgg aaaatttgtg acattgagtt tttggaataa ctgggaatgc tggataactc 2821 tttgggtaaa gctatggaac aaaagtacca gcatgaacta agaaatgact gtcacagcac 2881 aacatcaaac acaatgcaag acaaaggttc tctgcttttg ccttacttga cagaaaaagt 2941 aagctctagg ttcatcgcat gcagacagca gcaatacatg acatatataa aggctggctc 3001 ttttctccct tccattgaac tgaaacttcc actcagtcag acttgcgggg gatccacagc 3061 ctgactaact cgacaaaccc ccccaaacct aaggtgagtt gatctttaag ctttttacat 3121 tttcagctcg catatatcaa ttcgcgcgca ttgtcacact agcacccact agcggtcaga 3181 ctgcagattg cagcacgaaa caggaagctg actccacatg gtcacatgct cactgaagtg 3241 ttgacttccc tgacagctgt gcactttcta aaccggtttt ctcattcatt tacagttcag 3301 ccggaattca cctcgagacc atggctccaa agaagaagcg taaggtaacc atggcctcct 3361 ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg 3421 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg 3481 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc 3541 agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga 3601 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg 3661 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc 3721 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg 3781 aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga 3841 ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca 3901 agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc 3961 acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg 4021 gcgcctaaag ctgtgatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc 4081 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 4141 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 4201 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 4261 ggcttctgag gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc 4321 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 4381 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 4441 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt 4501 ggtcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg 4561 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa 4621 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta 4681 aagcaagtaa aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag 4741 cgatgcatgg gagctctgac gtacgttgtc tagcaaggaa gtgaagagct atccggatat 4801 atatgtatgt tatcataact gtactggcta tgtcaggtgg taatgactgt aacgttacgt 4861 tactcgttgt aggcacgaca ttgaatatgc tcttctccgc tgtggcccga acgcctcccg 4921 ctagcgatgg gatctaatac gactcactat agggagacca agggaaaata gaatgaagtg 4981 atctccaaaa aataagtact ttttgactgt aaataaaatt gtaaggagta aaaagtactt 5041 ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa 5101 gtaaagtaaa aatccccaaa aataatactt aagtacagta atcaagtaaa attactcaag 5161 tactttacac ctctgggatc cagctgcatc atgaacaata aaactgtctg cttacataaa 5221 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa 5281 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc 5341 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca 5401 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac 5461 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt 5521 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc 5581 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt 5641 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat 5701 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga 5761 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca 5821 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga 5881 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct 5941 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc 6001 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt 6061 taattggttg taacactggc aaaaaggatc taggtgaaga tcctttttga taatctcatg 6121 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 6181 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 6241 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 6301 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 6361 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 6421 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 6481 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 6541 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 6601 gttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 6661 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 6721 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 6781 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 6841 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 6901 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 6961 cagattt //