LOCUS       sequence-367447-        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-454 tNGFR-P2A-CARD11
FEATURES             Location/Qualifiers
     source          1..4398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..61)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     161..180
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     374..396
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     388..405
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     389..405
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1685..1741
                     /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(2187..2203)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2187..2203)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2200..2222)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2211..2227
                     /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(2235..2241,2242..2259,2260..2265))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2280..2301
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2418..2435)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2589..3177)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2669..2688)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3348..4139,4140..4208))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPIAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3971..3990
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4209..4313)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(4381..4398,1)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
       61 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      121 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      181 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
      241 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
      301 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
      361 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt
      421 acccggggac aaggctgcca tcttccctcc tccccatctc caggggtgtc aggacctgtg
      481 tcagtggcct cccaggctgt ccactcccca gggtggaaca gaagcccctc tccccctgca
      541 gcccctggtg tggtccactc tgccagtgtc atcgagggtg acacgcccct caggacttcc
      601 tggccttttc ttccaaagca gaacatctct ttgatcccag aacgattccc tttctctcca
      661 tcctccgcct cctgctccaa ggctgattat ctcttccttt tcagatccca agcactgcaa
      721 gtccagatgc aacgggagcc tggctcaagg gacgacaaga tccagccgga aagtgtagaa
      781 gtcacacccc aatggcggga tagcagcccc tgtgtgtgag cacccctcca tgggggcagg
      841 tgccactggc cgcgccatgg acgggccgcg cctgctgctg ttgctgcttc tgggggtgtc
      901 ccttggaggt gccaaggagg catgccccac aggcctgtac acacacagcg gtgagtgctg
      961 caaagcctgc aacctgggcg agggtgtggc ccagccttgt ggagccaacc agaccgtgtg
     1021 tgagccctgc ctggacagcg tgacgttctc cgacgtggtg agcgcgaccg agccgtgcaa
     1081 gccgtgcacc gagtgcgtgg ggctccagag catgtcggcg ccatgcgtgg aggccgacga
     1141 cgccgtgtgc cgctgcgcct acggctacta ccaggatgag acgactgggc gctgcgaggc
     1201 gtgccgcgtg tgcgaggcgg gctcgggcct cgtgttctcc tgccaggaca agcagaacac
     1261 cgtgtgcgag gagtgccccg acggcacgta ttccgacgag gccaaccacg tggacccgtg
     1321 cctgccctgc accgtgtgcg aggacaccga gcgccagctc cgcgagtgca cacgctgggc
     1381 cgacgccgag tgcgaggaga tccctggccg ttggattaca cggtccacac ccccagaggg
     1441 ctcggacagc acagccccca gcacccagga gcctgaggca cctccagaac aagacctcat
     1501 agccagcacg gtggcaggtg tggtgaccac agtgatgggc agctcccagc ccgtggtgac
     1561 ccgaggcacc accgacaacc tcatccctgt ctattgctcc atcctggctg ctgtggttgt
     1621 gggtcttgtg gcctacatag ccttcaagag gtggaacagc cgcgccaagc gctcgggttc
     1681 gggtgccacc aacttcagcc tgctgaagca ggccggcgac gtggaggaga accccggccc
     1741 cccaggtact cggcatctgt tatcccctct atacctctct atatattatg gagagacagg
     1801 gtcttgttct atcactgcgg ctggagtgca gtgatgtgat cataacatac tgcagccctc
     1861 cacctccttg gctcaagtga tcctcccatt ttagcctccc aagtagctgg gaccacaggc
     1921 acacacaacc aaatctggtg aattttttaa attttttgta gagacagggc cttgctccgt
     1981 tgcccagact ggtctcaaac tcctggcctc aagcgatctc cctgtctctg cctcccaaag
     2041 cactgattac aggcaggaga cactgtgcct ggccgttcct ttatattgga aaaaacgcaa
     2101 gtgatggtga cctgagactt ggcggagggg cgggagtagt gtcctctaga gtcgacctgc
     2161 aggcatgcaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2221 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2281 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2341 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgtgtatt
     2401 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2461 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2521 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2581 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2641 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2701 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2761 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2821 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2881 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2941 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3001 tggtggcata actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3061 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3121 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3181 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3241 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3301 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3361 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3421 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3481 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3541 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3601 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3661 gctataggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3721 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3781 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3841 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3901 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3961 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4021 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4081 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4141 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4201 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4261 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4321 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     4381 aataggcgta tcacgagg
//
