LOCUS       sequence-210649-        4160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA sequence source for amplifying an HDR template to tag endogenous
            human CD4 gene with BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4160)
  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 4160)
  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-BFP HDRT Source (pTR 176)
FEATURES             Location/Qualifiers
     source          1..4160
                     /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..1468
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     primer_bind     complement(1939..1955)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1939..1955)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1952..1974)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1963..1979
                     /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(1987..1993,1994..2011,2012..2017))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2032..2053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2341..2929)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2421..2440)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3100..3891,3892..3960))
                     /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     3723..3742
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3961..4065)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4133..4151
                     /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 gcagcgagct
      781 gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt
      841 caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa
      901 ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta
      961 cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt
     1021 ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc
     1081 tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt
     1141 gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac
     1201 cgagacgctg taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct
     1261 cgtgggcggg agccatctga tcgcaaacat caagaccaca tatagatcca agaaacccgc
     1321 taagaacctc aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga
     1381 ggccaacaac gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct
     1441 ccctagcaaa ctggggcaca agcttaatgg aaccggtgct ggaagtggtt gaggcacgag
     1501 gccaggcaga tcccacttgc agcctcccca ggtgtctgcc ccgcgtttcc tgcctgcgga
     1561 ccagatgaat gtagcagatc cccagcctct ggcctcctgt tcgcctcctc tacaatttgc
     1621 cattgtttct cctgggttag gccccggctt cactggttga gtgttgctct ctagtttcca
     1681 gaggcttaat cacaccgtcc tccacgccat ttccttttcc ttcaagccta gcccttctct
     1741 cattatttct ctctgaccct ctccccactg ctcatttgga tcccagggga gtgttcaggg
     1801 ccagccctgg ctggcatgga gggtgaggct gggtgtctgg aagcatcctg ctcacttgtt
     1861 tgccctcagg ctcgagtcct cgctcactga ctctcctcta gagtcgacct gcaggcatgc
     1921 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     1981 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2041 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2101 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2161 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2221 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2281 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2341 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2401 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2461 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2521 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2581 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2641 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2701 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2761 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     2821 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2881 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2941 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3001 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3061 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3121 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3181 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3241 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3301 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3361 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3421 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     3481 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3541 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3601 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3661 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3721 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3781 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3841 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3901 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3961 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4021 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4081 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     4141 tatcacgagg ccctttcgtc
//
