LOCUS Exported 4151 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION DNA sequence source for amplifying an HDR template to tag endogenous human CD4 gene with GFP. ACCESSION . VERSION . KEYWORDS CD4-C-GFP HDRT Source (pTR 152) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4151) 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 4151) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4151 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 364..386 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 378..395 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 379..395 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 475..493 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 773..1231 /codon_start=1 /product="improved N-terminal fragment of mVenus for use in bimolecular fluorescence complementation (BiFC) (Kodama and Hu, 2010)" /label=VN155(I152L) /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITA" primer_bind complement(812..833) /label=EGFP-N /note="EGFP, reverse primer" primer_bind 1420..1441 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1930..1946) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1930..1946) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1943..1965) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1954..1970 /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(1978..2008) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2023..2044 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2161..2178) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2332..2920) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2412..2431) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3091..3951) /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 3714..3733 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3952..4056) /gene="bla" /label=AmpR promoter primer_bind 4124..4142 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggag 421 tactgagagt gcaccaggat ccgggaacct cttctgtaac tccttagcag ctgataatac 481 gactcactat aggagatggg ggctaaaggg gtgtggtgga gaggatagag gggtgggaaa 541 agatggccag gagctagaag gaggcagaag tgggaggatg gagctgaagg agcagcaggc 601 caggaaaggc cctgctggaa agccactgga gctgtgctgc gctggaaagg ccattggagg 661 tgctagaacg caaaggggtt gcagtgggga cagacctgct ccccttcttc tttgttcctg 721 cagccggttt cagaagacat gtagccccat tggatcgggt gggactagtg gcagcaaggg 781 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg 841 ccacaagttc agcgtgcgcg gcgagggcga gggcgatgcc accaacggca agctgaccct 901 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct 961 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcgcc acgacttctt 1021 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcagcttca aggacgacgg 1081 cacctacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga 1141 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa 1201 cttcaacagc cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa 1261 cttcaagatc cgccacaacg tggaggacgg cagcgtgcag ctcgccgacc actaccagca 1321 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca 1381 gtccgtgctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt 1441 gaccgccgcc gggatcactg gaaccggtgc tggaagtggt tgaggcacga ggccaggcag 1501 atcccacttg cagcctcccc aggtgtctgc cccgcgtttc ctgcctgcgg accagatgaa 1561 tgtagcagat ccccagcctc tggcctcctg ttcgcctcct ctacaatttg ccattgtttc 1621 tcctgggtta ggccccggct tcactggttg agtgttgctc tctagtttcc agaggcttaa 1681 tcacaccgtc ctccacgcca tttccttttc cttcaagcct agcccttctc tcattatttc 1741 tctctgaccc tctccccact gctcatttgg atcccagggg agtgttcagg gccagccctg 1801 gctggcatgg agggtgaggc tgggtgtctg gaagcatcct gctcacttgt ttgccctcag 1861 gctcgagtcc tcgctcactg actctcctct agagtcgacc tgcaggcatg caagcttggc 1921 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 1981 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 2041 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 2101 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 2161 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 2221 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 2281 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 2341 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 2401 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 2461 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 2521 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 2581 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 2641 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 2701 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 2761 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 2821 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 2881 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 2941 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 3001 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 3061 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 3121 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 3181 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 3241 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 3301 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 3361 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 3421 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 3481 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 3541 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 3601 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 3661 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 3721 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 3781 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 3841 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 3901 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 3961 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 4021 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 4081 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 4141 gccctttcgt c //