LOCUS Exported 6796 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Donor for precise CRISPR directed genomic integration using the GeneWeld method. ACCESSION . VERSION . KEYWORDS pPRISM-gal4-VP16-cmlc2-mRFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6796) 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 6796) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6796 /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..998 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS VKASRRRGRKPRSF" 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 1000..1437 /codon_start=1 /gene="Saccharomyces cerevisiae GAL4 (truncated)" /product="DNA binding domain of the GAL4 transcriptional activator" /label=GAL4 DNA binding domain /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS" CDS 3413..3433 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3437..4114 /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(3567..3585) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 3835..3854 /label=mCherry-F /note="mCherry, forward primer" primer_bind 4029..4052 /label=DsRed1-C /note="DsRed1, forward primer" polyA_signal 4124..4348 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" polyA_signal 4418..4499 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 4623..4704 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter 5021..5039 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5315..5334) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 5326..6135 /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(5393..5412) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 6278..70 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6767..6786 /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 cccggagcca cgaacttctc tctgttaaag 961 caagcaggag acgtggaaga aaaccccggt ccttctagaa agctactgtc ttctatcgaa 1021 caagcatgcg atatttgccg acttaaaaag ctcaagtgct ccaaagaaaa accgaagtgc 1081 gccaagtgtc tgaagaacaa ctgggagtgt cgctactctc ccaaaaccaa aaggtctccg 1141 ctgactaggg cacatctgac agaagtggaa tcaaggctag aaagactgga acagctattt 1201 ctactgattt ttcctcgaga agaccttgac atgattttga aaatggattc tttacaggat 1261 ataaaagcat tgttaacagg attatttgta caagataatg tgaataaaga tgccgtcaca 1321 gatagattgg cttcagtgga gactgatatg cctctaacat tgagacagca tagaataagt 1381 gcgacatcat catcggaaga gagtagtaac aaaggtcaaa gacagttgac tgtatcgtcg 1441 aggtcgaccc cgggaataca gatctctcga gccgcccccc cgaccgatgt cagcctgggg 1501 gacgagctcc acttagacgg cgaggacgtg gcgatggcgc atgccgacgc gctagacgat 1561 ttcgatctgg acatgttggg ggacggggat tccccgggtc cgggatttac cccccacgac 1621 tccgccccct acggcgctct ggatatgtaa aactagtaat taagtctcag ccaccgttaa 1681 ctgaacatgt caaaacctgt ggagactgtt gagatttgat gttctgaaaa gataaagcct 1741 ataaataaaa tgttgcccaa atttcctgcc tgatgttttt ctttgtcttt gctacatggc 1801 tttgctgctc ggatcggctc actctgtgta tgccacgttc actttgtact ctccttctca 1861 cggtaggttt attattttta gatgtgcagt tagtttctgt gaaataacac accacacact 1921 gatattgtct gtgcattgac ttggtgagtg cacattgttt ttgatcttga catatttata 1981 tttgattgat caggtgaact gtgtgaatct aaagtgctcc atacagatgt tctgcattga 2041 aaatattctc attttattag tggaagtgag tgtatgccac atccaatcaa tttcagcaaa 2101 caccccagta tgatttaatg caaaaaaatg aaggtatcaa acacgcatta ctactttgca 2161 gttaaatatt taacatttat tccaacacga aaaaaagcag taaataacac tttgacaaac 2221 acgtcaggac atcttatttt tgtcaccctc acaggcaatt tagtataata tattatatat 2281 atatatatat catataataa tattcagtat aatatatata tatatatcat attataatat 2341 tcagtataat ataaaacaca aacacatata tgtataatat aatataacat ttttatttat 2401 tgagatgcct ctatggaccg tgttataaga agtaaagatc aggagaagta aacatgaagt 2461 gtaattatga atactgatgt taaattaagc tatgatgagt tttcactgtt aatttaccat 2521 ctcaattaaa tgttgatgcc tccatgacca agttaagcag atgagactga ggacaactgt 2581 agaagacaag atgttcactt tgctgaatat agctggcttg acagttatct atgactctat 2641 aaatatatat atattttttt tttataaaat gatttattta taactatata tccatttctc 2701 agacaggtgc ttcatatccc tcactcccgt agctgtccat gctggatctg tccccgttgt 2761 ttttaaaaag ctaaataagt tattaacatg actgcatcca gcgagccaaa cctgtctggt 2821 gtacagctac cagagaagct tgagatccta gtcaccggcg gcattcatcc atccttttca 2881 tccctcaaat ctctcattca cgtccccctc cccatctgca cactttatct cattttccac 2941 cctgctggaa tctgagcact tgtgcagtta tcagggctcc tgtatttagg aggctctggg 3001 tgtccatgta ggggacgaac agaaacactg cagaccttta tagaagaaca aatgataaga 3061 gtcctcatac ataaagactc cattagaaac gtcagtgacc caggagccca gaccaacagc 3121 aaagcagaca gtgaaggatc cacagcctga ctaactcgac aaaccccccc aaacctaagg 3181 tgagttgatc tttaagcttt ttacattttc agctcgcata tatcaattcg cgcgcattgt 3241 cacactagca cccactagcg gtcagactgc agattgcagc acgaaacagg aagctgactc 3301 cacatggtca catgctcact gaagtgttga cttccctgac agctgtgcac tttctaaacc 3361 ggttttctca ttcatttaca gttcagccgg aattcacctc gagaccatgg ctccaaagaa 3421 gaagcgtaag gtaaccatgg cctcctccga ggacgtcatc aaggagttca tgcgcttcaa 3481 ggtgcgcatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg 3541 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc 3601 cttcgcctgg gacatcctgt cccctcagtt ccagtacggc tccaaggcct acgtgaagca 3661 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg 3721 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga 3781 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt 3841 aatgcagaag aagaccatgg gctgggaggc ctccaccgag cggatgtacc ccgaggacgg 3901 cgccctgaag ggcgagatca agatgaggct gaagctgaag gacggcggcc actacgacgc 3961 cgaggtcaag accacctaca tggccaagaa gcccgtgcag ctgcccggcg cctacaagac 4021 cgacatcaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga 4081 gcgcgccgag ggccgccact ccaccggcgc ctaaagctgt gatctgtgcc ttctagttgc 4141 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 4201 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 4261 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 4321 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccaa agggcgaatt 4381 aattcattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt 4441 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 4501 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt 4561 aaagcaagta aaacctctac aaatgtggtc attgatgagt ttggacaaac cacaactaga 4621 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 4681 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 4741 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtcattca 4801 gatcaattaa ccctcacccg ggctagcgat gcatgggagc tctgacgtac gttgtctagc 4861 aaggaagtga agagctatcc ggatatatat gtatgttatc ataactgtac tggctatgtc 4921 aggtggtaat gactgtaacg ttacgttact cgttgtaggc acgacattga atatgctctt 4981 ctccgctgtg gcccgaacgc ctcccgctag cgatgggatc taatacgact cactataggg 5041 agaccaaggg aaaatagaat gaagtgatct ccaaaaaata agtacttttt gactgtaaat 5101 aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa 5161 aagtattgat ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt 5221 acagtaatca agtaaaatta ctcaagtact ttacacctct gggatccagc tgcatcatga 5281 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa 5341 cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa 5401 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc 5461 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat 5521 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt 5581 atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc 5641 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc 5701 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt 5761 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat 5821 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca 5881 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac 5941 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag 6001 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt 6061 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc 6121 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcaaaa aggatctagg 6181 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 6241 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 6301 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 6361 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 6421 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 6481 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 6541 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 6601 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 6661 agcgtgagct atgagaaagc gccacggttc ccgaagggag aaaggcggac aggtatccgg 6721 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 6781 atctttatag tcctgt //