LOCUS       sequence-367431-        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-458 tNGFR-P2A-STAT2
FEATURES             Location/Qualifiers
     source          1..4398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(28..50)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     150..169
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     363..385
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     377..394
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     378..394
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1674..1730
                     /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(2176..2192)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2176..2192)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2189..2211)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2200..2216
                     /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(2224..2230,2231..2248,2249..2254))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2269..2290
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2407..2424)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2578..3166)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2658..2677)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3337..4128,4129..4197))
                     /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     3960..3979
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4198..4302)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4370..4388
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
       61 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
      121 tggcgggtgt cgggactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
      181 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
      241 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
      301 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
      361 ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta cccggggaat
      421 ttcctttgag tgtcatgtgg gtgctcaaaa agttagattt tggaccattt cagatttcag
      481 agttttggat tagggatgct cgactggtaa gtaatgcaga tattccaaaa tctggacaaa
      541 tctgaaatcc aaaatgcttg gaatagcaga tactcaactg gtagcactcc ctggaagaat
      601 atgcaccaaa ctgatagcag tggttacttc tggagaggag gggaaagaac caagattagc
      661 agtaggatca acatatattt taatgttttc tgtattttta ttacttgtat aatttaaaca
      721 ttttaaatta gtaataatga acaatcatga aactatggat gatttagtcc agcaaaatat
      781 ccaattggga accctcatcc ttctgacgca gagcccaaat gggggcaggt gccactggcc
      841 gcgccatgga cgggccgcgc ctgctgctgt tgctgcttct gggggtgtcc cttggaggtg
      901 ccaaggaggc atgccccaca ggcctgtaca cacacagcgg tgagtgctgc aaagcctgca
      961 acctgggcga gggtgtggcc cagccttgtg gagccaacca gaccgtgtgt gagccctgcc
     1021 tggacagcgt gacgttctcc gacgtggtga gcgcgaccga gccgtgcaag ccgtgcaccg
     1081 agtgcgtggg gctccagagc atgtcggcgc catgcgtgga ggccgacgac gccgtgtgcc
     1141 gctgcgccta cggctactac caggatgaga cgactgggcg ctgcgaggcg tgccgcgtgt
     1201 gcgaggcggg ctcgggcctc gtgttctcct gccaggacaa gcagaacacc gtgtgcgagg
     1261 agtgccccga cggcacgtat tccgacgagg ccaaccacgt ggacccgtgc ctgccctgca
     1321 ccgtgtgcga ggacaccgag cgccagctcc gcgagtgcac acgctgggcc gacgccgagt
     1381 gcgaggagat ccctggccgt tggattacac ggtccacacc cccagagggc tcggacagca
     1441 cagcccccag cacccaggag cctgaggcac ctccagaaca agacctcata gccagcacgg
     1501 tggcaggtgt ggtgaccaca gtgatgggca gctcccagcc cgtggtgacc cgaggcacca
     1561 ccgacaacct catccctgtc tattgctcca tcctggctgc tgtggttgtg ggtcttgtgg
     1621 cctacatagc cttcaagagg tggaacagcc gcgccaagcg ctcgggttcg ggtgccacca
     1681 acttcagcct gctgaagcag gccggcgacg tggaggagaa ccccggcccc gcgcagtggg
     1741 aaatgctgca gaatcttgac agcccctttc aggatcagct gcaccagctt tactcgcaca
     1801 gcctcctgcc tgtggacatt cgacagtact tggctgtctg gattgaagac cagaactggt
     1861 gaggccttca ggaagttggg ggaatgaaaa aggtggttcc ttacttctgg gcccccggga
     1921 tcctggaatc attaatggca ggaaggggtt ggaaagcctc aggactacag taacactgca
     1981 gagacactaa tacttcttat tcctggtgcc caggcaggaa gctgcacttg ggagtgatga
     2041 ttccaaggct accatgctat tcttccactt cttggatcag ctgaactatg agtgtggccg
     2101 ttgcagccag gacccagagt ccttgttgct tcctctagag tcgacctgca ggcatgcaag
     2161 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2221 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2281 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2341 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2401 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2461 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2521 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2581 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2641 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2701 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2761 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2821 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2881 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2941 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3001 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3061 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3121 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3181 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3241 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3301 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3361 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3421 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3481 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3541 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3601 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3661 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3721 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3781 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     3841 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     3901 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     3961 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4021 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4081 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4141 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4201 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4261 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4321 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     4381 cacgaggccc tttcgtct
//
