LOCUS       sequence-210648-        4169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA sequence source for amplifying an HDR template to tag endogenous
            human CD4 gene with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4169)
  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 4169)
  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-mCherry HDRT Source (pTR 175)
FEATURES             Location/Qualifiers
     source          1..4169
                     /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..1477
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(915..933)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1183..1202
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1948..1964)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1948..1964)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1961..1983)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1972..1988
                     /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(1996..2002,2003..2020,2021..2026))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2041..2062
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2350..2938)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2430..2449)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3109..3900,3901..3969))
                     /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     3732..3751
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3970..4074)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4142..4160
                     /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 gcgtgagcaa
      781 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
      841 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
      901 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
      961 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     1021 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     1081 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     1141 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     1201 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     1261 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     1321 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     1381 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     1441 ccgccactcc accggcggca tggacgagct gtacaaggga accggtgctg gaagtggttg
     1501 aggcacgagg ccaggcagat cccacttgca gcctccccag gtgtctgccc cgcgtttcct
     1561 gcctgcggac cagatgaatg tagcagatcc ccagcctctg gcctcctgtt cgcctcctct
     1621 acaatttgcc attgtttctc ctgggttagg ccccggcttc actggttgag tgttgctctc
     1681 tagtttccag aggcttaatc acaccgtcct ccacgccatt tccttttcct tcaagcctag
     1741 cccttctctc attatttctc tctgaccctc tccccactgc tcatttggat cccaggggag
     1801 tgttcagggc cagccctggc tggcatggag ggtgaggctg ggtgtctgga agcatcctgc
     1861 tcacttgttt gccctcaggc tcgagtcctc gctcactgac tctcctctag agtcgacctg
     1921 caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     1981 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2041 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2101 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2161 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2221 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2281 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2341 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2401 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2461 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2521 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2581 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2641 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2701 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2761 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     2821 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2881 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2941 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3001 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3061 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3121 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3181 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     3241 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3301 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3361 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3421 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     3481 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     3541 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     3601 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     3661 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     3721 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     3781 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     3841 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     3901 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     3961 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4021 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4081 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     4141 aaataggcgt atcacgaggc cctttcgtc
//
