LOCUS Exported 3759 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Universal Cleavable Donor Plasmid for tagging with tdTomato using NHEJ. ACCESSION . VERSION . KEYWORDS pCRISPR-HOT_tdTomato SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3759) AUTHORS Artegiani B, Hendriks D, Beumer J, Kok R, Zheng X, Joore I, Chuva de Sousa Lopes S, van Zon J, Tans S, Clevers H TITLE Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing. JOURNAL Nat Cell Biol. 2020 Mar;22(3):321-331. doi: 10.1038/s41556-020-0472-5. Epub 2020 Mar 2. PUBMED 32123335 REFERENCE 2 (bases 1 to 3759) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3759 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 415..434 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(653..757) /gene="bla" /label=AmpR promoter primer_bind 952..974 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 966..983 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 967..983 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 1065..2492 /codon_start=1 /product="tandem dimeric (pseudo-monomeric) derivative of DsRed (Shaner et al., 2004)" /label=tdTomato /note="mammalian codon-optimized" /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH LFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYE GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVL KGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYER SEGRHHLFLYGMDELYK" primer_bind complement(2511..2528) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2517..2741 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin complement(2792..3380) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2872..2891) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3551..652) /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" ORIGIN 1 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 61 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 121 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 181 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 241 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 301 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 361 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 421 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 481 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 541 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 601 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 661 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 721 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgcggt 781 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca 841 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg 901 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 961 gacgttgtaa aacgacggcc agtgaattcg agctcggtac ctaatgatag ctagcgggcc 1021 agtacccaaa aagcgggggg tctggtggca gtggaggggg atccgtgagc aagggcgagg 1081 aggtcatcaa agagttcatg cgcttcaagg tgcgcatgga gggctccatg aacggccacg 1141 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc 1201 tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttca 1261 tgtacggctc caaggcgtac gtgaagcacc ccgccgacat ccccgattac aagaagctgt 1321 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggtctggtga 1381 ccgtgaccca ggactcctcc ctgcaggacg gcacgctgat ctacaaggtg aagatgcgcg 1441 gcaccaactt cccccccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct 1501 ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagatccac caggccctga 1561 agctgaagga cggcggccac tacctggtgg agttcaagac catctacatg gccaagaagc 1621 ccgtgcaact gcccggctac tactacgtgg acaccaagct ggacatcacc tcccacaacg 1681 aggactacac catcgtggaa cagtacgagc gctccgaggg ccgccaccac ctgttcctgg 1741 ggcatggcac cggcagcacc ggcagcggca gctccggcac cgcctcctcc gaggacaaca 1801 acatggccgt catcaaagag ttcatgcgct tcaaggtgcg catggagggc tccatgaacg 1861 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg 1921 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc 1981 agttcatgta cggctccaag gcgtacgtga agcaccccgc cgacatcccc gattacaaga 2041 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggtc 2101 tggtgaccgt gacccaggac tcctccctgc aggacggcac gctgatctac aaggtgaaga 2161 tgcgcggcac caacttcccc cccgacggcc ccgtaatgca gaagaagacc atgggctggg 2221 aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag atccaccagg 2281 ccctgaagct gaaggacggc ggccactacc tggtggagtt caagaccatc tacatggcca 2341 agaagcccgt gcaactgccc ggctactact acgtggacac caagctggac atcacctccc 2401 acaacgagga ctacaccatc gtggaacagt acgagcgctc cgagggccgc caccacctgt 2461 tcctgtacgg catggacgag ctgtacaagt aagcggccgc cgctgatcag cctcgactgt 2521 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 2581 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 2641 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 2701 agacaatagc aggcatgctg gggatgcggt gggctctatg gacgcgtcag caaaaggcca 2761 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 2821 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 2881 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 2941 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 3001 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 3061 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 3121 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 3181 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 3241 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 3301 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 3361 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 3421 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 3481 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 3541 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 3601 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 3661 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 3721 cagcaataaa ccagccagcc ggaagggccg agcgcagaa //