LOCUS       sequence-367294-        4407 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 4407)
  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 4407)
  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-374 tNGFR-P2A-B2M
FEATURES             Location/Qualifiers
     source          1..4407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(41..63)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     163..182
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     376..398
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     390..407
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     391..407
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1696..1752
                     /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(2198..2214)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2198..2214)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2211..2233)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2222..2238
                     /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(2246..2252,2253..2270,2271..2276))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2291..2312
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2429..2446)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2600..3188)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2680..2699)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3359..4150,4151..4219))
                     /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     3982..4001
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4220..4324)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(4392..4407,1..3)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
       61 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
      121 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
      181 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
      241 gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
      301 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
      361 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg
      421 gtacccgggg aaaaatgcag gtccgagcag ttaactggct ggggcaccat tagcaagtca
      481 cttagcatct ctggggccag tctgcaaagc gagggggcag ccttaatgtg cctccagcct
      541 gaagtcctag aatgagcgcc cggtgtccca agctggggcg cgcaccccag atcggagggc
      601 gccgatgtac agacagcaaa ctcacccagt ctagtgcatg ccttcttaaa catcacgaga
      661 ctctaagaaa aggaaactga aaacgggaaa gtccctctct ctaacctggc actgcgtcgc
      721 tggcttggag acaggtgacg gtccctgcgg gccttgtcct gattggctgg gcacgcgttt
      781 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag
      841 atgggggcag gtgccactgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt
      901 ctgggggtgt cccttggagg tgccaaggag gcatgcccca caggcctgta cacacacagc
      961 ggtgagtgct gcaaagcctg caacctgggc gagggtgtgg cccagccttg tggagccaac
     1021 cagaccgtgt gtgagccctg cctggacagc gtgacgttct ccgacgtggt gagcgcgacc
     1081 gagccgtgca agccgtgcac cgagtgcgtg gggctccaga gcatgtcggc gccatgcgtg
     1141 gaggccgacg acgccgtgtg ccgctgcgcc tacggctact accaggatga gacgactggg
     1201 cgctgcgagg cgtgccgcgt gtgcgaggcg ggctcgggcc tcgtgttctc ctgccaggac
     1261 aagcagaaca ccgtgtgcga ggagtgcccc gacggcacgt attccgacga ggccaaccac
     1321 gtggacccgt gcctgccctg caccgtgtgc gaggacaccg agcgccagct ccgcgagtgc
     1381 acacgctggg ccgacgccga gtgcgaggag atccctggcc gttggattac acggtccaca
     1441 cccccagagg gctcggacag cacagccccc agcacccagg agcctgaggc acctccagaa
     1501 caagacctca tagccagcac ggtggcaggt gtggtgacca cagtgatggg cagctcccag
     1561 cccgtggtga cccgaggcac caccgacaac ctcatccctg tctattgctc catcctggct
     1621 gctgtggttg tgggtcttgt ggcctacata gccttcaaga ggtggaacag ccgcgccaag
     1681 cgctcgggtt cgggtgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag
     1741 aaccccggcc cctctcgctc cgtcgcctta gctgtgctcg cgctactctc tctttctggc
     1801 ctggaggcta tccagcgtga gtctctccta ccctcccgct ctggtccttc ctctcccgct
     1861 ctgcaccctc tgtggccctc gctgtgctct ctcgctccgt gacttccctt ctccaagttc
     1921 tccttggtgg cccgccgtgg ggctagtcca gggctggatc tcggggaagc ggcggggtgg
     1981 cctgggagtg gggaaggggg tgcgcacccg ggacgcgcgc tacttgcccc tttcggcggg
     2041 gagcagggga gacctttggc ctacggcgac gggagggtcg ggacaaagtt tagggcgtcg
     2101 ataagcgtca gagcgccgag gttgggggag ggtttctctt ccgctctttc gctcctctag
     2161 agtcgacctg caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2221 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2281 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2341 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2401 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2461 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2521 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2581 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2641 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2701 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2761 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2821 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2881 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2941 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3001 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3061 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3121 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3181 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3241 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3301 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3361 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3421 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3481 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3541 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3601 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3661 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3721 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3781 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3841 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3901 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3961 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4021 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4081 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4141 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4201 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4261 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4321 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     4381 taacctataa aaataggcgt atcacga
//
