LOCUS Exported 6802 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Donor for precise CRISPR directed genomic integration using the GeneWeld method. ACCESSION . VERSION . KEYWORDS pPRISM-Fuse-cmlc2-TagBFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6802) 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 6802) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6802 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 224..241 /label=L4440 /note="L4440 vector, forward primer" protein_bind 532..553 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 568..598 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 606..622 /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 611..633 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 630..646 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 630..646 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 642..944 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRRR" primer_bind 665..685 /label=T3 /note="T3 promoter, forward primer" promoter 667..685 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter complement(733..751) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(761..778) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(761..777) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(770..792) /label=M13/pUC Forward /note="In lacZ gene" CDS 958..1632 /codon_start=1 /product="monomeric derivative of DsRed (Campbell et al., 2002)" /label=mRFP1 /note="mammalian codon-optimized" /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST GA" primer_bind complement(1085..1103) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1353..1372 /label=mCherry-F /note="mCherry, forward primer" primer_bind 1547..1570 /label=DsRed1-C /note="DsRed1, forward primer" CDS 3395..3415 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3419..4120 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" polyA_signal 4130..4354 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" polyA_signal 4424..4505 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 4629..4710 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter 5027..5045 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5321..5340) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 5332..6141 /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(5399..5418) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 6284..70 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6773..6792 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 61 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 121 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 181 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 241 ggaagcggaa cagatttaaa tggtaccgag cagaggtgta aaaagtactc aaaaatttta 301 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg 361 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt 421 aaggaagtaa aagtaaaagc aagaaagaaa actagagtgg tctcccttta gtgagggtca 481 attgatggga ggcgttcggg ccacagcgga cacgcaggtg cgcaacgcaa ttaatgtgag 541 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 601 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 661 gcgcgcaatt aaccctcact aaagggaaca aaagctggag gacctcgagg gggggcccgg 721 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac 781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 901 gcctgaatgg cgaatggacc tgccacactt ctaggaggag gtagcggagg tagcggagcc 961 tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg 1021 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag 1081 accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc 1141 cctcagttcc agtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac 1201 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc 1261 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg 1321 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc 1381 tgggaggcct ccaccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag 1441 atgaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac cacctacatg 1501 gccaagaagc ccgtgcagct gcccggcgcc tacaagaccg acatcaagct ggacatcacc 1561 tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg ccgccactcc 1621 accggagcct aagactagta attaagtctc agccaccgtt aactgaacat gtcaaaacct 1681 gtggagactg ttgagatttg atgttctgaa aagataaagc ctataaataa aatgttgccc 1741 aaatttcctg cctgatgttt ttctttgtct ttgctacatg gctttgctgc tcggatcggc 1801 tcactctgtg tatgccacgt tcactttgta ctctccttct cacggtaggt ttattatttt 1861 tagatgtgca gttagtttct gtgaaataac acaccacaca ctgatattgt ctgtgcattg 1921 acttggtgag tgcacattgt ttttgatctt gacatattta tatttgattg atcaggtgaa 1981 ctgtgtgaat ctaaagtgct ccatacagat gttctgcatt gaaaatattc tcattttatt 2041 agtggaagtg agtgtatgcc acatccaatc aatttcagca aacaccccag tatgatttaa 2101 tgcaaaaaaa tgaaggtatc aaacacgcat tactactttg cagttaaata tttaacattt 2161 attccaacac gaaaaaaagc agtaaataac actttgacaa acacgtcagg acatcttatt 2221 tttgtcaccc tcacaggcaa tttagtataa tatattatat atatatatat atcatataat 2281 aatattcagt ataatatata tatatatatc atattataat attcagtata atataaaaca 2341 caaacacata tatgtataat ataatataac atttttattt attgagatgc ctctatggac 2401 cgtgttataa gaagtaaaga tcaggagaag taaacatgaa gtgtaattat gaatactgat 2461 gttaaattaa gctatgatga gttttcactg ttaatttacc atctcaatta aatgttgatg 2521 cctccatgac caagttaagc agatgagact gaggacaact gtagaagaca agatgttcac 2581 tttgctgaat atagctggct tgacagttat ctatgactct ataaatatat atatattttt 2641 tttttataaa atgatttatt tataactata tatccatttc tcagacaggt gcttcatatc 2701 cctcactccc gtagctgtcc atgctggatc tgtccccgtt gtttttaaaa agctaaataa 2761 gttattaaca tgactgcatc cagcgagcca aacctgtctg gtgtacagct accagagaag 2821 cttgagatcc tagtcaccgg cggcattcat ccatcctttt catccctcaa atctctcatt 2881 cacgtccccc tccccatctg cacactttat ctcattttcc accctgctgg aatctgagca 2941 cttgtgcagt tatcagggct cctgtattta ggaggctctg ggtgtccatg taggggacga 3001 acagaaacac tgcagacctt tatagaagaa caaatgataa gagtcctcat acataaagac 3061 tccattagaa acgtcagtga cccaggagcc cagaccaaca gcaaagcaga cagtgaagga 3121 tccacagcct gactaactcg acaaaccccc ccaaacctaa ggtgagttga tctttaagct 3181 ttttacattt tcagctcgca tatatcaatt cgcgcgcatt gtcacactag cacccactag 3241 cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca 3301 ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta 3361 cagttcagcc ggaattcacc tcgagaccat ggctccaaag aagaagcgta aggtaaccat 3421 gagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa 3481 ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat 3541 gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag 3601 cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa 3661 gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt 3721 gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat 3781 cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga 3841 ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc 3901 cctgaagctc gtgggcggga gccatctgat cgcaaacatc aagaccacat atagatccaa 3961 gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag 4021 aatcaaggag gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata 4081 ctgcgacctc cctagcaaac tggggcacaa gctcaattaa agctgagatc tgtgccttct 4141 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 4201 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt 4261 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat 4321 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccaaaggg 4381 cgaattaatt cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 4441 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 4501 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg 4561 ttttttaaag caagtaaaac ctctacaaat gtggtcattg atgagtttgg acaaaccaca 4621 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt 4681 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt 4741 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt 4801 cattcagatc aattaaccct cacccgggct agcgatgcat gggagctctg acgtacgttg 4861 tctagcaagg aagtgaagag ctatccggat atatatgtat gttatcataa ctgtactggc 4921 tatgtcaggt ggtaatgact gtaacgttac gttactcgtt gtaggcacga cattgaatat 4981 gctcttctcc gctgtggccc gaacgcctcc cgctagcgat gggatctaat acgactcact 5041 atagggagac caagggaaaa tagaatgaag tgatctccaa aaaataagta ctttttgact 5101 gtaaataaaa ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt aattaagtaa 5161 aagtaaaagt attgattttt aattgtactc aagtaaagta aaaatcccca aaaataatac 5221 ttaagtacag taatcaagta aaattactca agtactttac acctctggga tccagctgca 5281 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat 5341 attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg 5401 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg 5461 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt 5521 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag 5581 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca 5641 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca 5701 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc 5761 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat 5821 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt 5881 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt 5941 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga 6001 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa 6061 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg 6121 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcaaaaagga 6181 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 6241 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 6301 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 6361 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 6421 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 6481 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 6541 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 6601 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 6661 acctacagcg tgagctatga gaaagcgcca cggttcccga agggagaaag gcggacaggt 6721 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 6781 cctggtatct ttatagtcct gt //