LOCUS Exported 5934 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Tp63 reporter targeting repair template. ACCESSION . VERSION . KEYWORDS TP63-T2A-Rox-EF1a-Rox-Venus-NLS repair template SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5934) TITLE A functional genetic toolbox for human tissue-derived organoids JOURNAL Unpublished REFERENCE 2 (bases 1 to 5934) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5934 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 1..589 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 490..509 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 743..760 /label=L4440 /note="L4440 vector, forward primer" protein_bind 877..898 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 913..943 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 951..967 /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 956..978 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 975..991 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 975..991 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS complement(1928..1948) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1949..2662) /codon_start=1 /product="yellow fluorescent protein (YFP) with fast and efficient maturation (Nagai et al., 2002)" /label=Venus /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(1991..2012) /label=EGFP-C /note="EGFP, forward primer" primer_bind 2340..2359 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2599..2620 /label=EGFP-N /note="EGFP, reverse primer" LTR 2732..3000 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" promoter complement(3013..3224) /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS complement(3296..3349) /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" primer_bind complement(4309..4326) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4309..4325) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4318..4340) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(4534..4553) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4653..4675 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4713..4731) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4799..4903 /gene="bla" /label=AmpR promoter CDS 4904..5764 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5122..5141) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt 841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta 901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg 961 ataacaattt cacacaggaa acagctatga ccaatattgt aagataaggt atctggtatt 1021 aaatttaagg gggttactga tatgctcaaa tacatgacgt cgggtgtttt tgaactggct 1081 gctatgggcc ttttaagaat tttcctcatt cccaaccaaa aactaaagag gacgtttgga 1141 catgaacttc ttcttataac tggattaaat caccccaacg tgaaccatca tcactagaag 1201 aaatgtcagt aatataaaat ataccaacac tgagaaaaga atacatatct acagtcttac 1261 ctacataata agcaatggaa atggatctac tcaaaggttt gatgtggcat gtatttagaa 1321 tccaaagaac tgaaagaatc tgtacatcat cgtcactcac atgtagcatt aagctctaga 1381 tacctatcac catacatgat accccttaag ggaaggagta tagacaagcc atccaaaaca 1441 agtaaattaa aaaatggtca ataagtgcaa ctcaattttc tttcatttaa attacatttt 1501 aaacaatact caatgccccc cacccaaaaa tctatactgt atatttatac atttatatat 1561 atagtcttag actataaggg tgaacatgct ttataaaagt caatgagaaa tctctgcagc 1621 ttaaggagac acccaactct tttttgctca cttctaccta gccatggcag ctacagttct 1681 gcaagctata gttttgaagg cttaaagcca gaatcagaat tagatgccag gtcagatgtt 1741 aggtaagagg tagcctctta cttctccttc ccctaagaaa tcagacaaga ggaaggggag 1801 gagctaggga gaaggctttg aagattaagc aggagtgctt ttagggggct ggcagttagg 1861 agagggatag gaagagctca catggtgagg ctcactcccc ctcctctttg atgcgctgtt 1921 gcttttatac ctttctcttc ttttttggct tgtacagctc gtccatgccg agagtgatcc 1981 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc 2041 tcagggcgga ctggtagctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga 2101 tgggggtgtt ctgctggtag tggtcggcga gctgcacgcc gccgtcctcg atgttgtggc 2161 ggatcttgaa gttggccttg atgccgttct tctgcttgtc ggcggtgata tagacgttgt 2221 ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga 2281 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg 2341 tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca 2401 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcgggcg aagcactgca 2461 ggccgtagcc cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc 2521 agatcagctt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc 2581 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga 2641 acagctcctc gcccttgctc acggatccta actttaaata attggcatta tttaaagtta 2701 tcatggtggc gtaggcgccg gtcacagctt ggatctgtaa cggcgcagaa cagaaaacga 2761 aacaaagacg tagagttgag caagcagggt caggcaaagc gtggagagcc ggctgagtct 2821 aggtaggctc caagggagcg ccggacaaag gcccggtctc gacctgagct ttaaacttac 2881 ctagacggcg gacgcagttc aggaggcacc acaggcggga ggcggcagaa cgcgactcaa 2941 ccggcgtgga tggcggcctc aggtagggcg gcgggcgcgt gaaggagaga tgcgagcccc 3001 tcgaagcttc agctgtgttc tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac 3061 tactgcactt atatacggtt ctcccccacc ctcgggaaaa aggcggagcc agtacacgac 3121 atcactttcc cagtttaccc cgcgccacct tctctaggca cccgttcaat tgccgacccc 3181 tccccccaac ttctcgggga ctgtgggcga tgtgcgctct gcccactgac gggcaccgga 3241 gcgatcgcag atcctttaac tttaaataat tggcattatt taaagttata ccggtagggc 3301 cgggattctc ctccacgtca ccgcatgtta gaagacttcc tctgccctct ccggatcctt 3361 cgccctcttc cttaattctc tgctgtttat tgcgtcgagc atccatgtca aagttgaagt 3421 cattccactc atctcggggt gggaaagaga tggtctggcg gagggtgaat cgcacagcat 3481 caataacacg ctcaccccgg gtctcactgg agcccacact gactgtagag gcactgcttg 3541 gggtccgcag gagatgagaa ggggaggaga attcgtggag ctgccggtgg tccaggatgc 3601 ccttccagat cgcatgtcga aattgctcag ggattttcag acttgccaga tcctgtgcaa 3661 caggggaagg tgtcatggag aatgaaaaca gggagaaaaa cggacattta gtccacagca 3721 tcctatcatt ccctgatgct ctggtttaat tgatctgaat ctattgatcc acaaattaga 3781 aaataaaatt agttctctga gtcttcaact ctatggaact aagcacaaag gcacagcctt 3841 gaggtgtctt tcctgcctgt gtagaacact tcacttagtc tctactggcc tcagtttctt 3901 cattggtaaa attgggataa tagctagatg agagggcaaa aatcccaaat agaaacatga 3961 ggtacctact tatgcttgaa tttacctggc tagtcactag aagaaaggta ttaatggaat 4021 gtcaccacca cttggttgag gagatgagaa gaaaagattg agtactttct atgtgccagg 4081 ccctgaatta ttttatattc atgatatcat ttaatcccca gattagtggt aaacattatt 4141 agacctgttt tatagataga acaactgaag gccagagagg ttgacaagtt taaagtcaca 4201 taactaagtg aaaaaattag ggacttaggg catcctcaga acctgaaacg gcacttttat 4261 ctaaaaggtc tcttggtgtt tgaataaagc aaggtatcaa aatgcagaac tggccgtcgt 4321 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 4381 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 4441 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg 4501 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 4561 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 4621 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 4681 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 4741 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 4801 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 4861 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 4921 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 4981 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 5041 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 5101 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 5161 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 5221 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 5281 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 5341 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 5401 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 5461 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 5521 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 5581 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 5641 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 5701 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 5761 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 5821 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 5881 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc //