LOCUS Exported 4169 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION DNA sequence source for amplifying an HDR template to tag endogenous human CD4 gene with mCherry. ACCESSION . VERSION . KEYWORDS CD4-C-mCherry HDRT Source (pTR 175) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4169) AUTHORS Roth TL, Puig-Saus C, Yu R, Shifrut E, Carnevale J, Li PJ, Hiatt J, Saco J, Krystofinski P, Li H, Tobin V, Nguyen DN, Lee MR, Putnam AL, Ferris AL, Chen JW, Schickel JN, Pellerin L, Carmody D, Alkorta-Aranburu G, Del Gaudio D, Matsumoto H, Morell M, Mao Y, Cho M, Quadros RM, Gurumurthy CB, Smith B, Haugwitz M, Hughes SH, Weissman JS, Schumann K, Esensten JH, May AP, Ashworth A, Kupfer GM, Greeley SAW, Bacchetta R, Meffre E, Roncarolo MG, Romberg N, Herold KC, Ribas A, Leonetti MD, Marson A TITLE Reprogramming human T cell function and specificity with non-viral genome targeting. JOURNAL Nature. 2018 Jul 11. pii: 10.1038/s41586-018-0326-5. doi: 10.1038/s41586-018-0326-5. PUBMED 29995861 REFERENCE 2 (bases 1 to 4169) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4169 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 42..60 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(98..120) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 220..239 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 433..455 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 447..464 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 448..464 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 544..562 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 842..1546 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(984..1002) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1252..1271 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(2017..2033) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2017..2033) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2030..2052) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2041..2057 /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)." promoter complement(2065..2095) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2110..2131 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2248..2265) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2419..3007) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2499..2518) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3178..4038) /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 3801..3820 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4039..4143) /gene="bla" /label=AmpR promoter ORIGIN 1 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 61 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 121 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 181 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac 241 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 301 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 361 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 421 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta 481 cccggggagt actgagagtg caccaggatc cgggaacctc ttctgtaact ccttagcagc 541 tgataatacg actcactata ggagatgggg gctaaagggg tgtggtggag aggatagagg 601 ggtgggaaaa gatggccagg agctagaagg aggcagaagt gggaggatgg agctgaagga 661 gcagcaggcc aggaaaggcc ctgctggaaa gccactggag ctgtgctgcg ctggaaaggc 721 cattggaggt gctagaacgc aaaggggttg cagtggggac agacctgctc cccttcttct 781 ttgttcctgc agccggtttc agaagacatg tagccccatt ggatcgggtg ggactagtgg 841 cgtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt 901 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg 961 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt 1021 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc 1081 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt 1141 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg 1201 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat 1261 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc 1321 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga 1381 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa 1441 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg 1501 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagggaa ccggtgctgg 1561 aagtggttga ggcacgaggc caggcagatc ccacttgcag cctccccagg tgtctgcccc 1621 gcgtttcctg cctgcggacc agatgaatgt agcagatccc cagcctctgg cctcctgttc 1681 gcctcctcta caatttgcca ttgtttctcc tgggttaggc cccggcttca ctggttgagt 1741 gttgctctct agtttccaga ggcttaatca caccgtcctc cacgccattt ccttttcctt 1801 caagcctagc ccttctctca ttatttctct ctgaccctct ccccactgct catttggatc 1861 ccaggggagt gttcagggcc agccctggct ggcatggagg gtgaggctgg gtgtctggaa 1921 gcatcctgct cacttgtttg ccctcaggct cgagtcctcg ctcactgact ctcctctaga 1981 gtcgacctgc aggcatgcaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 2041 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 2101 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 2161 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 2221 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 2281 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 2341 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 2401 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 2461 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 2521 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 2581 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 2641 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 2701 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 2761 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 2821 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 2881 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 2941 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 3001 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 3061 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 3121 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 3181 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 3241 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 3301 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 3361 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 3421 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 3481 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 3541 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 3601 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 3661 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 3721 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 3781 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 3841 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 3901 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 3961 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 4021 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 4081 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 4141 gcgcacattt ccccgaaaag tgccacctg //