LOCUS       sequence-215591-        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     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..1533
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     primer_bind     complement(2004..2020)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2004..2020)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2017..2039)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2028..2044
                     /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(2052..2058,2059..2076,2077..2082))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2097..2118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2406..2994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2486..2505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3165..3956,3957..4025))
                     /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     3788..3807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4026..4130)
                     /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 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
      901 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
      961 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     1021 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     1081 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     1141 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     1201 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     1261 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     1321 aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa
     1381 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     1441 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     1501 gacctcccta gcaaactggg gcacaagctt aatggaaccg gtgctggaag tggttgaggc
     1561 acgaggccag gcagatccca cttgcagcct ccccaggtgt ctgccccgcg tttcctgcct
     1621 gcggaccaga tgaatgtagc agatccccag cctctggcct cctgttcgcc tcctctacaa
     1681 tttgccattg tttctcctgg gttaggcccc ggcttcactg gttgagtgtt gctctctagt
     1741 ttccagaggc ttaatcacac cgtcctccac gccatttcct tttccttcaa gcctagccct
     1801 tctctcatta tttctctctg accctctccc cactgctcat ttggatccca ggggagtgtt
     1861 cagggccagc cctggctggc atggagggtg aggctgggtg tctggaagca tcctgctcac
     1921 ttgtttgccc tcaggctcga gtcctcgctc actgactctc ctctagagtc gacctgcagg
     1981 catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2041 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     2101 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     2161 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2221 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2281 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2341 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2401 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2461 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2521 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2581 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2641 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2701 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2761 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2821 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2881 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2941 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3001 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3061 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3121 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3181 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3241 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3301 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3361 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3421 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3481 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3541 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3601 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3661 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3721 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3781 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3841 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3901 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3961 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4021 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4081 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4141 cgaaaagtgc cacctgacgt
//
