LOCUS       sequence-215908-        4731 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA sequence source for amplifying an HDR template to tag endogenous
            human CLTA gene with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4731)
  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 4731)
  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: CLTA-mCherry HDRT Source (pTR 177)
FEATURES             Location/Qualifiers
     source          1..4731
                     /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"
     primer_bind     475..494
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        475..493
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1142..1843
                     /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
                     GRHSTGGMDELY"
     primer_bind     complement(1284..1302)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1552..1571
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2510..2526)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2510..2526)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2523..2545)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2534..2550
                     /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(2558..2564,2565..2582,2583..2588))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2603..2624
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2912..3500)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2992..3011)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3671..4462,4463..4531))
                     /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     4294..4313
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4532..4636)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4704..4722
                     /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 agggaccctc aaatccataa aaggattcaa tcttcttctt cttttttttt
      541 aggattcaat cttctaataa tcccgggagg gggcgcaaga gcaggattct tgtttctatt
      601 ttacgaatga ggaaactgag gttcagaaag cctaagtggc atgcctcttc agtaatcggc
      661 caatcgtcgc tgccctaact tcacctcacc tcccagtgca gcactagagt cccctcaggg
      721 gtggccgtcc ctggtcagtc cagctccagc agacggggtg ggtaggacgc tccctatcag
      781 gcagcacttc cgcctcccgg ggcccgcgca gctcacctcc ctcacctccc gccctacccc
      841 agtcacgagt tgttttaggg ggaccgcccc tccacttgct gattgggtag ctcctgaacc
      901 attgttgtcc tctgattggt tgttcccttt tcggctctgc aacaccgcct agaccgaccg
      961 gatacacggg tagggcttcc gctttacccg tctccctcct ggcgcttgtc ctcctctccc
     1021 agtcggcacc acagcggtgg ctgccgggcg tggtgtcggt gggtcggttg gtttttgtct
     1081 caccgttggt gtccgtgccg ttcagttgcc cgccatggct ggatcgggtg ggactagtgg
     1141 cgtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     1201 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     1261 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     1321 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     1381 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     1441 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     1501 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     1561 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     1621 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     1681 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     1741 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     1801 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacatgggaa ccggtgctgg
     1861 aagtggtgag ctggatccgt tcggcgcccc tgccggcgcc cctggcggtc ccgcgctggg
     1921 gaacggagtg gccggcgccg gcgaagaaga cccggctgcg gccttcttgg cgcagcaaga
     1981 gagcgagatt gcgggcatcg agaacgacga ggccttcgcc atcctggacg gcggcgcccc
     2041 cgggccccag ccgcacggcg agccgccggg gggtccgggt gagagtgcgg gcgcgtttgg
     2101 ggcgagagga cttgtctgga aactcggtcc acagtgggtc cgagagcttc tgtgtgactc
     2161 gtgctccttg ctgaattagg aggttaggga gcagtgcaaa caggaaacga gaccctggcc
     2221 cggtctttca gaaacctagg ctcgagaagc ctgttcggtt ctcagcatgt ttgagtgctt
     2281 ctgggcgcgg gcggagcgag aaagcaagtg tagggtggca ggctccggag ccggaagaag
     2341 cccgttcaat tcagcaactt ttcattaagc atttgctgtg cctttagtcc gggcttccta
     2401 ctatcgtcag agacttccct gctcacttgt ttgccctcag gctcgagtcc tcgctcactg
     2461 actctcctct agagtcgacc tgcaggcatg caagcttggc gtaatcatgg tcatagctgt
     2521 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     2581 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2641 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2701 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2761 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2821 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2881 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2941 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3001 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3061 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3121 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3181 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3241 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3301 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3361 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3421 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3481 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3541 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3601 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3661 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3721 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3781 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3841 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3901 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3961 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4021 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4081 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4141 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4201 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4261 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4321 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4381 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4441 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4501 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4561 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4621 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     4681 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
