LOCUS       sequence-367331-        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-379 tNGFR-P2A-CD48
FEATURES             Location/Qualifiers
     source          1..4398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..53)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     153..172
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     366..388
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     380..397
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     381..397
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1677..1733
                     /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(2179..2195)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2179..2195)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2192..2214)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2203..2219
                     /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(2227..2233,2234..2251,2252..2257))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2272..2293
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2410..2427)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2581..3169)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2661..2680)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3340..4131,4132..4200))
                     /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     3963..3982
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4201..4305)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4373..4391
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
       61 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
      121 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
      181 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
      241 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
      301 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      361 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg gtacccgggg
      421 agggaagctc ctatgtctta gcccctgaat taatcagagg gattcctgcc tgggaacgaa
      481 aagcagacat tctctatcag ggtcccttat tcttttctga gtactggccc ccctgagagt
      541 acagtagaag ctatgaatct tccccctaca aaaaagcact catgtacctt caaacaaaat
      601 tgttctcagt ttccgtgatg ctcttagata aaaaaattta caagacctcc ggtttctggt
      661 ttctggggcc ccacacaagt gtgctgggtg gagagaaaat ctcagtggtt tttttttttt
      721 tctctctaac cctgtttggt aagttccgtt tttagccccg gcctttttct agccaggctc
      781 tcaactgtct cctgcgttgc tgggaagttc tggaaggaag catgggggca ggtgccactg
      841 gccgcgccat ggacgggccg cgcctgctgc tgttgctgct tctgggggtg tcccttggag
      901 gtgccaagga ggcatgcccc acaggcctgt acacacacag cggtgagtgc tgcaaagcct
      961 gcaacctggg cgagggtgtg gcccagcctt gtggagccaa ccagaccgtg tgtgagccct
     1021 gcctggacag cgtgacgttc tccgacgtgg tgagcgcgac cgagccgtgc aagccgtgca
     1081 ccgagtgcgt ggggctccag agcatgtcgg cgccatgcgt ggaggccgac gacgccgtgt
     1141 gccgctgcgc ctacggctac taccaggatg agacgactgg gcgctgcgag gcgtgccgcg
     1201 tgtgcgaggc gggctcgggc ctcgtgttct cctgccagga caagcagaac accgtgtgcg
     1261 aggagtgccc cgacggcacg tattccgacg aggccaacca cgtggacccg tgcctgccct
     1321 gcaccgtgtg cgaggacacc gagcgccagc tccgcgagtg cacacgctgg gccgacgccg
     1381 agtgcgagga gatccctggc cgttggatta cacggtccac acccccagag ggctcggaca
     1441 gcacagcccc cagcacccag gagcctgagg cacctccaga acaagacctc atagccagca
     1501 cggtggcagg tgtggtgacc acagtgatgg gcagctccca gcccgtggtg acccgaggca
     1561 ccaccgacaa cctcatccct gtctattgct ccatcctggc tgctgtggtt gtgggtcttg
     1621 tggcctacat agccttcaag aggtggaaca gccgcgccaa gcgctcgggt tcgggtgcca
     1681 ccaacttcag cctgctgaag caggccggcg acgtggagga gaaccccggc ccctgctcca
     1741 gaggttggga ttcgtgtctg gctctggaat tgctactgct gcctctgtca ctcctggtga
     1801 ccagcattca aggtactttc caccaactgt gtatctgtga ttgtgcactg aaggcatggt
     1861 cagttgggaa aggggagacg ggatgtagtt caaccctaga agccagatct ggtgtctgga
     1921 aagcaggtat ttccattcca atagcatggc catggcatgg ttggggagcc tttcttagca
     1981 ctgatgaatg gagcttcatc tcctttccca cggggatgtg atcaaaacag gaacctgggg
     2041 agcaagagaa gtaggctcct catattttgt cccgtggttg gtggtccatg aagttgtgga
     2101 aaagtgtttg ttttttcatt ttgaataaga agatcctcta gagtcgacct gcaggcatgc
     2161 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     2221 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2281 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2341 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2401 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2461 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2521 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2581 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2641 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2701 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2761 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2821 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2881 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2941 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3001 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3061 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3121 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3181 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3241 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3301 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3361 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3421 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3481 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3541 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3601 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3661 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     3721 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3781 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3841 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3901 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3961 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4021 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4081 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4141 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4201 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4261 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4321 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     4381 tatcacgagg ccctttcg
//
