LOCUS       sequence-367448-        4398 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockin of truncated NGFR to target gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4398)
  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 4398)
  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-455 tNGFR-P2A-WAS
FEATURES             Location/Qualifiers
     source          1..4398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(38..60)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     160..179
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     373..395
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     387..404
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     388..404
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1684..1740
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(2186..2202)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2186..2202)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2199..2221)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2210..2226
                     /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(2234..2240,2241..2258,2259..2264))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2279..2300
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2417..2434)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2588..3176)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2668..2687)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3347..4138,4139..4207))
                     /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     3970..3989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4208..4312)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4380..4398
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
       61 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
      121 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
      181 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
      241 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      301 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      361 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta
      421 cccggggaag gtgctttaga aaggaggcca cccaggccca tgactactcc ttgccacagg
      481 gagccctgca cacagatgtg ctaagctctc gctgccagcc agagggagga gggtctgagc
      541 cagtcagaag gagatgggcc ccagagagta agaaaggggg aggaggaccc aagctgatcc
      601 aaaaggtggg tctaagcagt caagtggagg agggttccaa tctgatggcg gagggcccaa
      661 gctcagccta acgaggaggc caggcccacc aaggggcccc tggaggactt gtttcccttg
      721 tcccttgtgg ttttttgcat ttcctgttcc cttgctgctc attgcggaag ttcctcttct
      781 taccctgcac ccagagcctc gccagagaag acaagggcag aaagcaccat gggggcaggt
      841 gccactggcc gcgccatgga cgggccgcgc ctgctgctgt tgctgcttct gggggtgtcc
      901 cttggaggtg ccaaggaggc atgccccaca ggcctgtaca cacacagcgg tgagtgctgc
      961 aaagcctgca acctgggcga gggtgtggcc cagccttgtg gagccaacca gaccgtgtgt
     1021 gagccctgcc tggacagcgt gacgttctcc gacgtggtga gcgcgaccga gccgtgcaag
     1081 ccgtgcaccg agtgcgtggg gctccagagc atgtcggcgc catgcgtgga ggccgacgac
     1141 gccgtgtgcc gctgcgccta cggctactac caggatgaga cgactgggcg ctgcgaggcg
     1201 tgccgcgtgt gcgaggcggg ctcgggcctc gtgttctcct gccaggacaa gcagaacacc
     1261 gtgtgcgagg agtgccccga cggcacgtat tccgacgagg ccaaccacgt ggacccgtgc
     1321 ctgccctgca ccgtgtgcga ggacaccgag cgccagctcc gcgagtgcac acgctgggcc
     1381 gacgccgagt gcgaggagat ccctggccgt tggattacac ggtccacacc cccagagggc
     1441 tcggacagca cagcccccag cacccaggag cctgaggcac ctccagaaca agacctcata
     1501 gccagcacgg tggcaggtgt ggtgaccaca gtgatgggca gctcccagcc cgtggtgacc
     1561 cgaggcacca ccgacaacct catccctgtc tattgctcca tcctggctgc tgtggttgtg
     1621 ggtcttgtgg cctacatagc cttcaagagg tggaacagcc gcgccaagcg ctcgggttcg
     1681 ggtgccacca acttcagcct gctgaagcag gccggcgacg tggaggagaa ccccggcccc
     1741 agtgggggcc caatgggagg aaggcccggg ggccgaggag caccagcggt tcagcagaac
     1801 ataccctcca ccctcctcca ggaccacgag aaccagcgac tctttgagat gcttggacga
     1861 aaatgcttgg tgagctgggg atctcctgcc cccgccccgt ccccaccgtt tcttcctctt
     1921 cctctcctcc ttctctctct tcccctcctc ccgctcctcc tttccctctc catcatctcc
     1981 tctcctagaa tttcccgtca taatccaccc ttcccaggaa gatctcaatg tctacttgcc
     2041 ttccctctgg ctgcagctct tcctttgggc ccatgactgt catgaggcag gaaggaccag
     2101 gtctggctcc aagaccttgt ggctacccct gaccagactc tcctctagag tcgacctgca
     2161 ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     2221 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     2281 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     2341 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2401 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2461 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2521 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2581 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2641 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2701 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2761 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2821 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2881 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2941 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3001 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3061 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3121 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3181 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3241 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3301 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3361 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3421 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3481 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3541 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3601 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3661 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3721 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3781 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3841 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3901 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3961 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4021 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4081 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4141 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4201 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4261 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     4321 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     4381 ataggcgtat cacgaggc
//
