LOCUS       sequence-367268-        4280 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 4280)
  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 4280)
  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-338 pUC19-tNGFR-P2A-LAG3 N
FEATURES             Location/Qualifiers
     source          1..4280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(34..56)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     156..175
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     369..391
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     383..400
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     384..400
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1627..1683
                     /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(2064..2080)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2064..2080)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2077..2099)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2088..2104
                     /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(2112..2118,2119..2136,2137..2142))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2157..2178
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2295..2312)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2466..3054)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2546..2565)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3225..4016,4017..4085))
                     /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     3848..3867
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4086..4190)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4258..4276
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
       61 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
      121 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
      181 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
      241 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
      301 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
      361 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcgagc tcggtacccg
      421 gggaaggggt gaaggcccag agaccagcag aacggcatcc cagccacgac ggccactttg
      481 ctctgtctgc tctccgccac ggccctgctc tgttccctgg gacacccccg cccccacctc
      541 ctcaggctgc ctgatctgcc cagctttcca gctttcctct ggattccggc ctctggtcat
      601 ccctccccac cctctctcca aggccctctc ctggtctccc ttcttctaga accccttcct
      661 ccacctccct ctctgcagaa cttctccttt accccccacc ccccaccact gccccctttc
      721 cttttctgac ctccttttgg agggctcagc gctgcccaga ccataggaga gatgggggca
      781 ggtgccactg gccgcgccat ggacgggccg cgcctgctgc tgttgctgct tctgggggtg
      841 tcccttggag gtgccaagga ggcatgcccc acaggcctgt acacacacag cggtgagtgc
      901 tgcaaagcct gcaacctggg cgagggtgtg gcccagcctt gtggagccaa ccagaccgtg
      961 tgtgagccct gcctggacag cgtgacgttc tccgacgtgg tgagcgcgac cgagccgtgc
     1021 aagccgtgca ccgagtgcgt ggggctccag agcatgtcgg cgccatgcgt ggaggccgac
     1081 gacgccgtgt gccgctgcgc ctacggctac taccaggatg agacgactgg gcgctgcgag
     1141 gcgtgccgcg tgtgcgaggc gggctcgggc ctcgtgttct cctgccagga caagcagaac
     1201 accgtgtgcg aggagtgccc cgacggcacg tattccgacg aggccaacca cgtggacccg
     1261 tgcctgccct gcaccgtgtg cgaggacacc gagcgccagc tccgcgagtg cacacgctgg
     1321 gccgacgccg agtgcgagga gatccctggc cgttggatta cacggtccac acccccagag
     1381 ggctcggaca gcacagcccc cagcacccag gagcctgagg cacctccaga acaagacctc
     1441 atagccagca cggtggcagg tgtggtgacc acagtgatgg gcagctccca gcccgtggtg
     1501 acccgaggca ccaccgacaa cctcatccct gtctattgct ccatcctggc tgctgtggtt
     1561 gtgggtcttg tggcctacat agccttcaag aggtggaaca gccgcgccaa gcgctcgggt
     1621 tcgggtgcca ccaacttcag cctgctgaag caggccggcg acgtggagga gaaccccggc
     1681 cccatgtggg aggctcagtt cctgggcttg ctgtttctgc agccgctttg ggtggctcca
     1741 ggtaaaacgg ggatggcggg agggttgacc tccagcccca caggagggga ccagcaggga
     1801 tctctgtggc cacaaaggtc ctgaggtcct tagctctgtg gattcttcta atcccttttt
     1861 tgggcagtcc ttccaccccg aaagcctctc tgggcagaga agaaacagaa acccaagttc
     1921 ttcctgcacc ctgtttctcc ctcgggaaac acccaggctc cttctctacc cctgcctctc
     1981 ggctcacgcc ccctcccctt ggcctctctt ttgctcactc ctctagagtc gacctgcagg
     2041 catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2101 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     2161 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     2221 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2281 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2341 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2401 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2461 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2521 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2581 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2641 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2701 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2761 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2821 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2881 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2941 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3001 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3061 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3121 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3181 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3241 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3301 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3361 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3421 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3481 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3541 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3601 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3661 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3721 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3781 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3841 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3901 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3961 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4021 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4081 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4141 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4201 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     4261 aggcgtatca cgaggccctt
//
