LOCUS       sequence-367324-        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-459 tNGFR-P2A-STAT6
FEATURES             Location/Qualifiers
     source          1..4398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(42..64)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     164..183
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     377..399
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     391..408
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     392..408
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1688..1744
                     /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(2190..2206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2190..2206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2203..2225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2214..2230
                     /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(2238..2244,2245..2262,2263..2268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2283..2304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2421..2438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2592..3180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2672..2691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3351..4142,4143..4211))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMRDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3974..3993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4212..4316)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(4384..4398,1..4)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
       61 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
      121 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
      181 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
      241 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
      301 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
      361 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagctc
      421 ggtacccggg gactaacact catattttat agatgagaag atcgaagccc acaaagggaa
      481 ggctctttgc ccacagaacc agagccaggt ctagagctgc aactaaatcc tctgccactc
      541 taagagagct ctcgctctac tgccctgtct ccctttgcct ccccatccct ctggctacag
      601 ctcagctctt cccacccctg tgtctatcac tgaaggagtt acccccatct caggcattga
      661 ctcaggatgc ccctggttta aggtggtctg gccatgagtg gtggtgggga cggtccctag
      721 gagggctatc tatgggaggt ccctggctgc cccaggagat aggccaagtt tcttgggcac
      781 ccctcagagt ggccttattt ttctcctcca ggcaacctcc aagtcccaga tcatgggggc
      841 aggtgccact ggccgcgcca tggacgggcc gcgcctgctg ctgttgctgc ttctgggggt
      901 gtcccttgga ggtgccaagg aggcatgccc cacaggcctg tacacacaca gcggtgagtg
      961 ctgcaaagcc tgcaacctgg gcgagggtgt ggcccagcct tgtggagcca accagaccgt
     1021 gtgtgagccc tgcctggaca gcgtgacgtt ctccgacgtg gtgagcgcga ccgagccgtg
     1081 caagccgtgc accgagtgcg tggggctcca gagcatgtcg gcgccatgcg tggaggccga
     1141 cgacgccgtg tgccgctgcg cctacggcta ctaccaggat gagacgactg ggcgctgcga
     1201 ggcgtgccgc gtgtgcgagg cgggctcggg cctcgtgttc tcctgccagg acaagcagaa
     1261 caccgtgtgc gaggagtgcc ccgacggcac gtattccgac gaggccaacc acgtggaccc
     1321 gtgcctgccc tgcaccgtgt gcgaggacac cgagcgccag ctccgcgagt gcacacgctg
     1381 ggccgacgcc gagtgcgagg agatccctgg ccgttggatt acacggtcca cacccccaga
     1441 gggctcggac agcacagccc ccagcaccca ggagcctgag gcacctccag aacaagacct
     1501 catagccagc acggtggcag gtgtggtgac cacagtgatg ggcagctccc agcccgtggt
     1561 gacccgaggc accaccgaca acctcatccc tgtctattgc tccatcctgg ctgctgtggt
     1621 tgtgggtctt gtggcctaca tagccttcaa gaggtggaac agccgcgcca agcgctcggg
     1681 ttcgggtgcc accaacttca gcctgctgaa gcaggccggc gacgtggagg agaaccccgg
     1741 cccctctctg tggggtctgg tctccaagat gcccccagaa aaagtgcagc ggctctatgt
     1801 cgactttccc caacacctgc ggcatcttct gggtgactgg ctggagagcc agccctggtg
     1861 agtcctggct gctccctgct ggtcccccaa gtcttcccta actcatcttc cttctcctta
     1921 gatttttctc ccctcaccca tggattcaga acttgagacc tgttattcca tgtgtagtga
     1981 cctagattta gcagggagtc tgtgccccat caagaccagg ctatgaatgt tgacagatgg
     2041 agaccccatc tcttaggagg ctgagccgaa gaggaggggg gtttgggctg ggacaaaggc
     2101 acttctcata acagctagaa gactgggaaa caaggcgcat gggttcctct agagtcgacc
     2161 tgcaggcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2221 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2281 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2341 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2401 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2461 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2521 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2581 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2641 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2701 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2761 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2821 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2881 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2941 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3001 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3061 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3121 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3181 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3241 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3301 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3361 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3421 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3481 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3541 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3601 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3661 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3721 tcccaacgat caaggcgagt tacatgatcc ctcatgttgt gcaaaaaagc ggttagctcc
     3781 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3841 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3901 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3961 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4021 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4081 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4141 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4201 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4261 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4321 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     4381 aaaaataggc gtatcacg
//
