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 38..56 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(94..116) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 216..235 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 429..451 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 443..460 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 444..460 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 540..558 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 838..1296 /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(877..898) /label=EGFP-N /note="EGFP, reverse primer" primer_bind 1485..1506 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1995..2011) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1995..2011) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2008..2030) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2019..2035 /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(2043..2073) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2088..2109 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2226..2243) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2397..2985) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2477..2496) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3156..4016) /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 3779..3798 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4017..4121) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt 61 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 121 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 181 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 241 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 301 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 361 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc 421 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcgagc tcggtacccg 481 gggagtactg agagtgcacc aggatccggg aacctcttct gtaactcctt agcagctgat 541 aatacgactc actataggag atgggggcta aaggggtgtg gtggagagga tagaggggtg 601 ggaaaagatg gccaggagct agaaggaggc agaagtggga ggatggagct gaaggagcag 661 caggccagga aaggccctgc tggaaagcca ctggagctgt gctgcgctgg aaaggccatt 721 ggaggtgcta gaacgcaaag gggttgcagt ggggacagac ctgctcccct tcttctttgt 781 tcctgcagcc ggtttcagaa gacatgtagc cccattggat cgggtgggac tagtggcagc 841 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 901 aacggccaca agttcagcgt gcgcggcgag ggcgagggcg atgccaccaa cggcaagctg 961 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 1021 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcgccacgac 1081 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatcag cttcaaggac 1141 gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 1201 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 1261 tacaacttca acagccacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag 1321 gccaacttca agatccgcca caacgtggag gacggcagcg tgcagctcgc cgaccactac 1381 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 1441 acccagtccg tgctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 1501 ttcgtgaccg ccgccgggat cactggaacc ggtgctggaa gtggttgagg cacgaggcca 1561 ggcagatccc acttgcagcc tccccaggtg tctgccccgc gtttcctgcc tgcggaccag 1621 atgaatgtag cagatcccca gcctctggcc tcctgttcgc ctcctctaca atttgccatt 1681 gtttctcctg ggttaggccc cggcttcact ggttgagtgt tgctctctag tttccagagg 1741 cttaatcaca ccgtcctcca cgccatttcc ttttccttca agcctagccc ttctctcatt 1801 atttctctct gaccctctcc ccactgctca tttggatccc aggggagtgt tcagggccag 1861 ccctggctgg catggagggt gaggctgggt gtctggaagc atcctgctca cttgtttgcc 1921 ctcaggctcg agtcctcgct cactgactct cctctagagt cgacctgcag gcatgcaagc 1981 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 2041 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 2101 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 2161 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 2221 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 2281 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 2341 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 2401 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 2461 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 2521 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 2581 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 2641 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 2701 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 2761 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 2821 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 2881 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 2941 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3001 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3061 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3121 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 3181 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 3241 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 3301 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 3361 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 3421 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 3481 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 3541 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 3601 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 3661 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 3721 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 3781 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 3841 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 3901 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 3961 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 4021 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 4081 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 4141 ccacctgacg t //