LOCUS       sequence-215587-        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     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..1350
                     /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(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(join(2043..2049,2050..2067,2068..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."
     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(join(3156..3947,3948..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
//
