LOCUS Exported 6833 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-eGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6833) 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 6833) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6833 /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..4156 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 3437..4153 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3482..3503) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3743..3762) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4090..4111 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 4161..4385 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" polyA_signal 4455..4536 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 4660..4741 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter 5058..5076 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5352..5371) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 5363..6172 /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(5430..5449) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 6315..70 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6804..6823 /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 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat 3481 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga 3541 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc 3601 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta 3661 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca 3721 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt 3781 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg 3841 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc 3901 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg 3961 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct 4021 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa 4081 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga 4141 cgagctgtac aagtaaagat ctgtgccttc tagttgccag ccatctgttg tttgcccctc 4201 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 4261 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 4321 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 4381 tatggcttct gaggcggaaa gaaccaaagg gcgaattaat tcattgatga gtttggacaa 4441 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 4501 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 4561 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa 4621 tgtggtcatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat 4681 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt 4741 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt 4801 ttaaagcaag taaaacctct acaaatgtgg tcattcagat caattaaccc tcacccgggc 4861 tagcgatgca tgggagctct gacgtacgtt gtctagcaag gaagtgaaga gctatccgga 4921 tatatatgta tgttatcata actgtactgg ctatgtcagg tggtaatgac tgtaacgtta 4981 cgttactcgt tgtaggcacg acattgaata tgctcttctc cgctgtggcc cgaacgcctc 5041 ccgctagcga tgggatctaa tacgactcac tatagggaga ccaagggaaa atagaatgaa 5101 gtgatctcca aaaaataagt actttttgac tgtaaataaa attgtaagga gtaaaaagta 5161 cttttttttc taaaaaaatg taattaagta aaagtaaaag tattgatttt taattgtact 5221 caagtaaagt aaaaatcccc aaaaataata cttaagtaca gtaatcaagt aaaattactc 5281 aagtacttta cacctctggg atccagctgc atcatgaaca ataaaactgt ctgcttacat 5341 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt 5401 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca 5461 atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa 5521 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct 5581 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg 5641 gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga 5701 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc 5761 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg 5821 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt 5881 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac 5941 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat 6001 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg 6061 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa 6121 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt 6181 ggttaattgg ttgtaacact ggcaaaaagg atctaggtga agatcctttt tgataatctc 6241 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 6301 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 6361 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 6421 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 6481 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 6541 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 6601 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 6661 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 6721 acggttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 6781 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgt //