LOCUS       sequence-367847-        4731 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knockin of mCherry to CLTA target.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4731)
  AUTHORS   Shy BR, Vykunta VS, Ha A, Talbot A, Roth TL, Nguyen DN, Pfeifer WG, 
            Chen YY, Blaeschke F, Shifrut E, Vedova S, Mamedov MR, Chung JJ, Li 
            H, Yu R, Wu D, Wolf J, Martin TG, Castro CE, Ye L, Esensten JH, 
            Eyquem J, Marson A
  TITLE     High-yield genome engineering in primary cells using a hybrid ssDNA 
            repair template and small-molecule cocktails.
  JOURNAL   Nat Biotechnol. 2022 Aug 25:10.1038/s41587-022-01418-8. doi: 
            10.1038/s41587-022-01418-8.
  PUBMED    36008610
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: "Nat 
            Biotechnol. 2022 Aug 25:10.1038/s41587-022-01418-8. doi: 
            10.1038/s41587-022-01418-8."
COMMENT     Sequence Label: pTR-177 pUC19-CLTA-N-mCherry
FEATURES             Location/Qualifiers
     source          1..4731
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(4..22)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        90..194
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(195..263,264..1055)
                     /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     complement(413..432)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1226..1814
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1715..1734
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2102..2123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2138..2143,2144..2161,2162..2168)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2176..2192
                     /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)."
     primer_bind     2181..2203
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2200..2216
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2200..2216
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(2883..3584)
                     /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(3155..3174)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3424..3442
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4232..4251)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4233..4251)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4331..4348)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4331..4347)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4340..4362)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4556..4575)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4675..4697
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
       61 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      121 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      181 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
      241 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
      301 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
      361 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
      421 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
      481 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
      541 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
      601 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
      661 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
      721 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
      781 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
      841 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
      901 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
      961 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1021 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1081 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1141 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1201 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     1261 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1321 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     1381 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1441 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1501 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1561 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     1621 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     1681 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     1741 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     1801 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     1861 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     1921 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     1981 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2041 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     2101 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     2161 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     2221 attacgccaa gcttgcatgc ctgcaggtcg actctagagg agagtcagtg agcgaggact
     2281 cgagcctgag ggcaaacaag tgagcaggga agtctctgac gatagtagga agcccggact
     2341 aaaggcacag caaatgctta atgaaaagtt gctgaattga acgggcttct tccggctccg
     2401 gagcctgcca ccctacactt gctttctcgc tccgcccgcg cccagaagca ctcaaacatg
     2461 ctgagaaccg aacaggcttc tcgagcctag gtttctgaaa gaccgggcca gggtctcgtt
     2521 tcctgtttgc actgctccct aacctcctaa ttcagcaagg agcacgagtc acacagaagc
     2581 tctcggaccc actgtggacc gagtttccag acaagtcctc tcgccccaaa cgcgcccgca
     2641 ctctcacccg gaccccccgg cggctcgccg tgcggctggg gcccgggggc gccgccgtcc
     2701 aggatggcga aggcctcgtc gttctcgatg cccgcaatct cgctctcttg ctgcgccaag
     2761 aaggccgcag ccgggtcttc ttcgccggcg ccggccactc cgttccccag cgcgggaccg
     2821 ccaggggcgc cggcaggggc gccgaacgga tccagctcac cacttccagc accggttccc
     2881 atgtacagct cgtccatgcc gccggtggag tggcggccct cggcgcgttc gtactgttcc
     2941 acgatggtgt agtcctcgtt gtgggaggtg atgtccaact tgatgttgac gttgtaggcg
     3001 ccgggcagct gcacgggctt cttggccttg taggtggtct tgacctcagc gtcgtagtgg
     3061 ccgccgtcct tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg
     3121 tacatccgct cggaggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg
     3181 gaggggaagt tggtgccgcg cagcttcacc ttgtagatga actcgccgtc ctgcagggag
     3241 gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg
     3301 aagccctcgg ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag
     3361 gccttggagc cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggcca
     3421 cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg
     3481 ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaagcgcatg
     3541 aactccttga tgatggccat gttatcctcc tcgcccttgc tcacgccact agtcccaccc
     3601 gatccagcca tggcgggcaa ctgaacggca cggacaccaa cggtgagaca aaaaccaacc
     3661 gacccaccga caccacgccc ggcagccacc gctgtggtgc cgactgggag aggaggacaa
     3721 gcgccaggag ggagacgggt aaagcggaag ccctacccgt gtatccggtc ggtctaggcg
     3781 gtgttgcaga gccgaaaagg gaacaaccaa tcagaggaca acaatggttc aggagctacc
     3841 caatcagcaa gtggaggggc ggtcccccta aaacaactcg tgactggggt agggcgggag
     3901 gtgagggagg tgagctgcgc gggccccggg aggcggaagt gctgcctgat agggagcgtc
     3961 ctacccaccc cgtctgctgg agctggactg accagggacg gccacccctg aggggactct
     4021 agtgctgcac tgggaggtga ggtgaagtta gggcagcgac gattggccga ttactgaaga
     4081 ggcatgccac ttaggctttc tgaacctcag tttcctcatt cgtaaaatag aaacaagaat
     4141 cctgctcttg cgccccctcc cgggattatt agaagattga atcctaaaaa aaaagaagaa
     4201 gaagattgaa tccttttatg gatttgaggg tccctatagt gagtcgtatt atcagctgct
     4261 aaggagttac agaagaggtt cccggatcct ggtgcactct cagtactccc cgggtaccga
     4321 gctcgaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     4381 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4441 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     4501 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     4561 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4621 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4681 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga a
//
