LOCUS       sequence-210647-        4151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA sequence source for amplifying an HDR template to tag endogenous
            human CD4 gene with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2018 Jul 11. pii: 10.1038/s41586-018-0326-5. doi: 
            10.1038/s41586-018-0326-5."
COMMENT     Sequence Label: CD4-C-GFP HDRT Source (pTR 152)
FEATURES             Location/Qualifiers
     source          1..4151
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..1285
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVE"
     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(join(1978..1984,1985..2002,2003..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."
     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(join(3091..3882,3883..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
//
