LOCUS       sequence-367266-        4498 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 4498)
  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 4498)
  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-337 pUC19-tNGFR-P2A-CTLA4 N
FEATURES             Location/Qualifiers
     source          1..4498
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(49..71)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     171..190
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     384..406
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     398..415
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     399..415
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1745..1801
                     /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(2297..2313)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2297..2313)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2310..2332)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2321..2337
                     /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(2345..2351,2352..2369,2370..2375))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2390..2411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2528..2545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2699..3287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2779..2798)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3458..4249,4250..4318))
                     /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     4081..4100
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4319..4423)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(4491..4498,1..11)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
       61 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      121 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      181 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
      241 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
      301 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      361 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
      421 cgagctcggt acccggggat ttggagttgt caatgaaatg aattggactg gatggttaag
      481 gatgcccaga agattgaata aaattgggat ttaggaggac ccttgtactc caggaaattc
      541 tccaagtctc cacttagtta tccagatcct caaagtgaac atgaagcttc agtttcaaat
      601 tgaatacatt ttccatccat ggattggctt gttttgttca gttgagtgct tgaggttgtc
      661 ttttcgacgt aacagctaaa cccacggctt cctttctcgt aaaaccaaaa caaaaaggct
      721 ttctattcaa gtgccttctg tgtgtgcaca tgtgtaatac atatctggga tcaaagctat
      781 ctatataaag tccttgattc tgtgtgggtt caaacacatt tcaaagcttc aggatcctga
      841 aaggttttgc tctacttcct gaagacctga acaccgctcc cataaagcca tgggggcagg
      901 tgccactggc cgcgccatgg acgggccgcg cctgctgctg ttgctgcttc tgggggtgtc
      961 ccttggaggt gccaaggagg catgccccac aggcctgtac acacacagcg gtgagtgctg
     1021 caaagcctgc aacctgggcg agggtgtggc ccagccttgt ggagccaacc agaccgtgtg
     1081 tgagccctgc ctggacagcg tgacgttctc cgacgtggtg agcgcgaccg agccgtgcaa
     1141 gccgtgcacc gagtgcgtgg ggctccagag catgtcggcg ccatgcgtgg aggccgacga
     1201 cgccgtgtgc cgctgcgcct acggctacta ccaggatgag acgactgggc gctgcgaggc
     1261 gtgccgcgtg tgcgaggcgg gctcgggcct cgtgttctcc tgccaggaca agcagaacac
     1321 cgtgtgcgag gagtgccccg acggcacgta ttccgacgag gccaaccacg tggacccgtg
     1381 cctgccctgc accgtgtgcg aggacaccga gcgccagctc cgcgagtgca cacgctgggc
     1441 cgacgccgag tgcgaggaga tccctggccg ttggattaca cggtccacac ccccagaggg
     1501 ctcggacagc acagccccca gcacccagga gcctgaggca cctccagaac aagacctcat
     1561 agccagcacg gtggcaggtg tggtgaccac agtgatgggc agctcccagc ccgtggtgac
     1621 ccgaggcacc accgacaacc tcatccctgt ctattgctcc atcctggctg ctgtggttgt
     1681 gggtcttgtg gcctacatag ccttcaagag gtggaacagc cgcgccaagc gctcgggttc
     1741 gggtgccacc aacttcagcc tgctgaagca ggccggcgac gtggaggaga accccggccc
     1801 catggcttgc cttggatttc agcggcacaa ggctcagctg aacctggcta ccaggacctg
     1861 gccctgcact ctcctgtttt ttcttctctt catccctgtc ttctgcaaag gtgagtgaga
     1921 cttttggagc atgaagatgg aggaggtgtt tctcctacct gggtttcatt tgtttcagca
     1981 gtcaaaggca gtgatttata gcaaagccag aagttaaagg taaaactcca atctggcttg
     2041 gctggctctg tattccaggg ccagcaggga gcagttgggc ggcagcaaat aaggcaaaga
     2101 gatagctcag aacagagcgc caggtattta gtaggggctt catgaatgca tgtgagttgg
     2161 tttagtagag agacacaggc aatttcagac ccttctatga gactggaagt gatttaagag
     2221 ggaaaggata gccatagtcc tgaatacatt ttcctctaga gtcgacctgc aggcatgcaa
     2281 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2341 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2401 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2461 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2521 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2581 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2641 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2701 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2761 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2821 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2881 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2941 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3001 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3061 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3121 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3181 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3241 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3301 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3361 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3421 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3481 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3541 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3601 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3661 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3721 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3781 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3841 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3901 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3961 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4021 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4081 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4141 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4201 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4261 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4321 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4381 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4441 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcg
//
