LOCUS Exported 7195 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Donor for precise CRISPR directed genomic integration using the GeneWeld method. ACCESSION . VERSION . KEYWORDS pPRISM-Cre-cmlc2-TagBFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7195) 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 7195) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7195 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 225..242 /label=L4440 /note="L4440 vector, forward primer" protein_bind 533..554 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 569..599 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 607..623 /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 612..634 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 631..647 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 631..647 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 643..1056 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS VKASRRRGRKPRSYVQFTDRTPKFACITGRCNE" primer_bind 666..686 /label=T3 /note="T3 promoter, forward primer" promoter 668..686 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter complement(734..752) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(762..779) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(762..778) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(771..793) /label=M13/pUC Forward /note="In lacZ gene" CDS 995..2026 /codon_start=1 /product="site-specific recombinase" /label=Cre /note="Cre is a site-specific recombinase from bacteriophage P1. Recombination occurs at loxP sequences." /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE DGD" CDS 3789..3809 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3813..4514 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" polyA_signal 4524..4748 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" polyA_signal 4818..4899 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 5023..5104 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter 5421..5439 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5715..5734) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 5726..6535 /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(5793..5812) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 6678..71 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7167..7186 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 61 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 121 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 181 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 241 aggaagcgga acagatttaa atggtaccga gcagaggtgt aaaaagtact caaaaatttt 301 actcaagtga aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct 361 ggctagaatc ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat 421 taaggaagta aaagtaaaag caagaaagaa aactagagtg gtctcccttt agtgagggtc 481 aattgatggg aggcgttcgg gccacagcgg acacgcaggt gcgcaacgca attaatgtga 541 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 601 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 661 agcgcgcaat taaccctcac taaagggaac aaaagctgga ggacctcgag ggggggcccg 721 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa 781 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 841 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 901 agcctgaatg gcgaatggac ctgccacact tcccggagcc acgaacttct ctctgttaaa 961 gcaagcagga gacgtggaag aaaaccccgg tcctatgtcc aatttactga ccgtacacca 1021 aaatttgcct gcattaccgg tcgatgcaac gagtgatgag gttcgcaaga acctgatgga 1081 catgttcagg gatcgccagg cgttttctga gcatacctgg aaaatgcttc tgtccgtttg 1141 ccggtcgtgg gcggcatggt gcaagttgaa taaccggaaa tggtttcccg cagaacctga 1201 agatgttcgc gattatcttc tatatcttca ggcgcgcggt ctggcagtaa aaactatcca 1261 gcaacatttg ggccagctaa acatgcttca tcgtcggtcc gggctgccac gaccaagtga 1321 cagcaatgct gtttcactgg ttatgcggcg gatccgaaaa gaaaacgttg atgccggtga 1381 acgtgcaaaa caggctctag cgttcgaacg cactgatttc gaccaggttc gttcactcat 1441 ggaaaatagc gatcgctgcc aggatatacg taatctggca tttctgggga ttgcttataa 1501 caccctgtta cgtatagccg aaattgccag gatcagggtt aaagatatct cacgtactga 1561 cggtgggaga atgttaatcc atattggcag aacgaaaacg ctggttagca ccgctggagt 1621 agagaaggca cttagcctgg gggtaactaa actggtcgag cgatggattt ccgtctctgg 1681 tgtagctgat gatccgaata actacctgtt ttgccgggtc agaaaaaatg gtgttgccgc 1741 gccatctgcc accagccagc tatcaactcg cgccctggaa gggatttttg aagcaactca 1801 tcgattgatt tacggcgcta aggatgactc tggtcagaga tacctggcct ggtctggaca 1861 cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct ggagtttcaa taccggagat 1921 catgcaagct ggtggctgga ccaatgtaaa tattgtcatg aactatatcc gtaacctgga 1981 tagtgaaaca ggggcaatgg tgcgcctgct ggaagatggc gattagaact agtaattaag 2041 tctcagccac cgttaactga acatgtcaaa acctgtggag actgttgaga tttgatgttc 2101 tgaaaagata aagcctataa ataaaatgtt gcccaaattt cctgcctgat gtttttcttt 2161 gtctttgcta catggctttg ctgctcggat cggctcactc tgtgtatgcc acgttcactt 2221 tgtactctcc ttctcacggt aggtttatta tttttagatg tgcagttagt ttctgtgaaa 2281 taacacacca cacactgata ttgtctgtgc attgacttgg tgagtgcaca ttgtttttga 2341 tcttgacata tttatatttg attgatcagg tgaactgtgt gaatctaaag tgctccatac 2401 agatgttctg cattgaaaat attctcattt tattagtgga agtgagtgta tgccacatcc 2461 aatcaatttc agcaaacacc ccagtatgat ttaatgcaaa aaaatgaagg tatcaaacac 2521 gcattactac tttgcagtta aatatttaac atttattcca acacgaaaaa aagcagtaaa 2581 taacactttg acaaacacgt caggacatct tatttttgtc accctcacag gcaatttagt 2641 ataatatatt atatatatat atatatcata taataatatt cagtataata tatatatata 2701 tatcatatta taatattcag tataatataa aacacaaaca catatatgta taatataata 2761 taacattttt atttattgag atgcctctat ggaccgtgtt ataagaagta aagatcagga 2821 gaagtaaaca tgaagtgtaa ttatgaatac tgatgttaaa ttaagctatg atgagttttc 2881 actgttaatt taccatctca attaaatgtt gatgcctcca tgaccaagtt aagcagatga 2941 gactgaggac aactgtagaa gacaagatgt tcactttgct gaatatagct ggcttgacag 3001 ttatctatga ctctataaat atatatatat ttttttttta taaaatgatt tatttataac 3061 tatatatcca tttctcagac aggtgcttca tatccctcac tcccgtagct gtccatgctg 3121 gatctgtccc cgttgttttt aaaaagctaa ataagttatt aacatgactg catccagcga 3181 gccaaacctg tctggtgtac agctaccaga gaagcttgag atcctagtca ccggcggcat 3241 tcatccatcc ttttcatccc tcaaatctct cattcacgtc cccctcccca tctgcacact 3301 ttatctcatt ttccaccctg ctggaatctg agcacttgtg cagttatcag ggctcctgta 3361 tttaggaggc tctgggtgtc catgtagggg acgaacagaa acactgcaga cctttataga 3421 agaacaaatg ataagagtcc tcatacataa agactccatt agaaacgtca gtgacccagg 3481 agcccagacc aacagcaaag cagacagtga aggatccaca gcctgactaa ctcgacaaac 3541 ccccccaaac ctaaggtgag ttgatcttta agctttttac attttcagct cgcatatatc 3601 aattcgcgcg cattgtcaca ctagcaccca ctagcggtca gactgcagat tgcagcacga 3661 aacaggaagc tgactccaca tggtcacatg ctcactgaag tgttgacttc cctgacagct 3721 gtgcactttc taaaccggtt ttctcattca tttacagttc agccggaatt cacctcgaga 3781 ccatggctcc aaagaagaag cgtaaggtaa ccatgagcga gctgattaag gagaacatgc 3841 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg 3901 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc 3961 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca 4021 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat 4081 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc 4141 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg 4201 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg 4261 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc 4321 tgatcgcaaa catcaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc 4381 ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct 4441 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc 4501 acaagctcaa ttaaagctga gatctgtgcc ttctagttgc cagccatctg ttgtttgccc 4561 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 4621 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 4681 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 4741 ctctatggct tctgaggcgg aaagaaccaa agggcgaatt aattcattga tgagtttgga 4801 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt 4861 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat 4921 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac 4981 aaatgtggtc attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 5041 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 5101 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt 5161 tttttaaagc aagtaaaacc tctacaaatg tggtcattca gatcaattaa ccctcacccg 5221 ggctagcgat gcatgggagc tctgacgtac gttgtctagc aaggaagtga agagctatcc 5281 ggatatatat gtatgttatc ataactgtac tggctatgtc aggtggtaat gactgtaacg 5341 ttacgttact cgttgtaggc acgacattga atatgctctt ctccgctgtg gcccgaacgc 5401 ctcccgctag cgatgggatc taatacgact cactataggg agaccaaggg aaaatagaat 5461 gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa 5521 gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt 5581 actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta 5641 ctcaagtact ttacacctct gggatccagc tgcatcatga acaataaaac tgtctgctta 5701 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg 5761 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg 5821 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct 5881 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg 5941 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc 6001 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc 6061 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat 6121 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc 6181 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc 6241 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt 6301 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg 6361 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa 6421 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga 6481 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga 6541 attggttaat tggttgtaac actggcaaaa aggatctagg tgaagatcct ttttgataat 6601 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 6661 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 6721 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 6781 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 6841 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 6901 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 6961 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 7021 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 7081 gccacggttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 7141 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctg //