LOCUS       sequence-367429-        4051 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 4051)
  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 4051)
  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-341 pUC19-tNGFR-P2A-PDCD1 N
FEATURES             Location/Qualifiers
     source          1..4051
                     /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             1538..1594
                     /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(1829..1845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1829..1845)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1842..1864)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1853..1869
                     /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(1877..1883,1884..1901,1902..1907))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1922..1943
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2060..2077)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2231..2819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2311..2330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2990..3781,3782..3850))
                     /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     3613..3632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3851..3955)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4023..4041
                     /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 cggggctggc 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 cccggggaac
      421 gggcccacct cctccccatc tcctctgtct ccctgtctct gtctctctct ccctccccca
      481 ccctctcccc agtcctaccc cctcctcacc cctcctcccc cagcactgcc tctgtcactc
      541 tcgcccacgt ggatgtggag gaagaggggg cgggagcaag gggcgggcac cctcccttca
      601 acctgacctg ggacagtttc ccttccgctc acctccgcct gagcagtgga gaaggcggca
      661 ctctggtggg gctgctccag gcatgggggc aggtgccact ggccgcgcca tggacgggcc
      721 gcgcctgctg ctgttgctgc ttctgggggt gtcccttgga ggtgccaagg aggcatgccc
      781 cacaggcctg tacacacaca gcggtgagtg ctgcaaagcc tgcaacctgg gcgagggtgt
      841 ggcccagcct tgtggagcca accagaccgt gtgtgagccc tgcctggaca gcgtgacgtt
      901 ctccgacgtg gtgagcgcga ccgagccgtg caagccgtgc accgagtgcg tggggctcca
      961 gagcatgtcg gcgccatgcg tggaggccga cgacgccgtg tgccgctgcg cctacggcta
     1021 ctaccaggat gagacgactg ggcgctgcga ggcgtgccgc gtgtgcgagg cgggctcggg
     1081 cctcgtgttc tcctgccagg acaagcagaa caccgtgtgc gaggagtgcc ccgacggcac
     1141 gtattccgac gaggccaacc acgtggaccc gtgcctgccc tgcaccgtgt gcgaggacac
     1201 cgagcgccag ctccgcgagt gcacacgctg ggccgacgcc gagtgcgagg agatccctgg
     1261 ccgttggatt acacggtcca cacccccaga gggctcggac agcacagccc ccagcaccca
     1321 ggagcctgag gcacctccag aacaagacct catagccagc acggtggcag gtgtggtgac
     1381 cacagtgatg ggcagctccc agcccgtggt gacccgaggc accaccgaca acctcatccc
     1441 tgtctattgc tccatcctgg ctgctgtggt tgtgggtctt gtggcctaca tagccttcaa
     1501 gaggtggaac agccgcgcca agcgctcggg ttcgggtgcc accaacttca gcctgctgaa
     1561 gcaggccggc gacgtggagg agaaccccgg ccccatgcag attccacaag cgccctggcc
     1621 agtcgtctgg gcggtgctac aactgggctg gcggccagga tggttcttag gtaggtgggg
     1681 tcggcggtca ggtgtcccag agccaggggt ctggagggac cttccaccct cagtccctgg
     1741 caggtcgggg ggtgctgagg cgggcctggc cctggcagcc cagtcctcta gagtcgacct
     1801 gcaggcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     1861 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     1921 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     1981 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2041 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2101 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2161 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2221 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2281 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2341 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2401 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2461 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2521 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2581 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2641 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     2701 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2761 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2821 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2881 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     2941 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3001 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3061 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3121 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3181 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3241 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3301 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3361 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3421 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3481 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3541 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3601 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3661 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3721 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3781 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     3841 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3901 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3961 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     4021 aaaataggcg tatcacgagg ccctttcgtc t
//
