LOCUS       sequence-367271-        4397 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 4397)
  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 4397)
  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-449 tNGFR-P2A-CCR2
FEATURES             Location/Qualifiers
     source          1..4397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(37..59)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     159..178
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     372..394
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     386..403
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     387..403
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1683..1739
                     /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(2184..2200)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2184..2200)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2197..2219)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2208..2224
                     /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(2232..2238,2239..2256,2257..2262))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2277..2298
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2415..2432)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2586..3174)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2666..2685)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3345..4136,4137..4205))
                     /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     3968..3987
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4206..4310)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4378..4396
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
       61 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
      121 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
      181 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
      241 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
      301 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
      361 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattcg agctcggtac
      421 ccggggaaag tgcatgaata ggaggggtga gcaactgtgt gtccatcacc tttttgtcaa
      481 agaagcagga gtcagtgggc tacgtgcttc atgagcagga gaggcggaaa ctaaggaagg
      541 ctcatgtgtt ggaggaagca tgtttgaaga gcagcaggtc tcacagagtt tgctctttaa
      601 tactctcccc agcacacgga aggggaaggg ggtggaggtt gctgctatga gagagaaaaa
      661 aaaaacagcc acaatagaga ttctgccttc aaaggttggc ttgccacctg aagcagccac
      721 tgcccagggg gtgcaaagaa gagacagcag cgcccagctt ggaggtgcta actccagagg
      781 ccagcatcag caactgggca cagaaaggag ccgcctgggc agggaccatg ggggcaggtg
      841 ccactggccg cgccatggac gggccgcgcc tgctgctgtt gctgcttctg ggggtgtccc
      901 ttggaggtgc caaggaggca tgccccacag gcctgtacac acacagcggt gagtgctgca
      961 aagcctgcaa cctgggcgag ggtgtggccc agccttgtgg agccaaccag accgtgtgtg
     1021 agccctgcct ggacagcgtg acgttctccg acgtggtgag cgcgaccgag ccgtgcaagc
     1081 cgtgcaccga gtgtgtgggg ctccagagca tgtcggcgcc atgcgtggag gccgacgacg
     1141 ccgtgtgccg ctgcgcctac ggctactacc aggatgagac gactgggcgc tgcgaggcgt
     1201 gccgcgtgtg cgaggcgggc tcgggcctcg tgttctcctg ccaggacaag cagaacaccg
     1261 tgtgcgagga gtgccccgac ggcacgtatt ccgacgaggc caaccacgtg gacccgtgcc
     1321 tgccctgcac cgtgtgcgag gacaccgagc gccagctccg cgagtgcaca cgctgggccg
     1381 acgccgagtg cgaggagatc cctggccgtt ggattacacg gtccacaccc ccagagggct
     1441 cggacagcac agcccccagc acccaggagc ctgaggcacc tccagaacaa gacctcatag
     1501 ccagcacggt ggcaggtgtg gtgaccacag tgatgggcag ctcccagccc gtggtgaccc
     1561 gaggcaccac cgacaacctc atccctgtct attgctccat cctggctgct gtggttgtgg
     1621 gtcttgtggc ctacatagcc ttcaagaggt ggaacagccg cgccaagcgc tcgggttcgg
     1681 gtgccaccaa cttcagcctg ctgaagcagg ccggcgacgt ggaggagaac cccggccccg
     1741 cacggccaca tccctggtgg ctgtgcgttc tggggaccct ggtggggctc tcagctactc
     1801 cagcccccaa gagctgccca gagaggcact actgggctca gggaaagctg tgctgccaga
     1861 tgtgtgagcc aggtaagagg gggccttggt aagggccagg tgagtggcga aagagagagg
     1921 actggggtta atacagtaaa taggcgcagg gtagactgag ctcaagcaag gagggaaatc
     1981 ctgcagctgt ggggaggcac caccttgaag agggcagaga accagccctt ctcaggcctt
     2041 gatcccttac cctctcctcc caggaacatt cctcgtgaag gactgtgacc agcatagaaa
     2101 ggctgctcag tgtgatcctt gcataccggg ggtctccttc ctctaaagtc gacctgcagg
     2161 catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2221 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     2281 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     2341 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2401 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2461 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2521 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2581 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2641 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2701 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2761 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2821 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2881 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2941 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3001 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3061 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3121 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3181 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3241 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3301 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3361 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3421 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3481 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3541 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3601 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3661 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3721 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3781 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3841 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3901 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3961 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4021 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4081 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4141 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4201 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4261 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4321 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     4381 aggcgtatca cgaggcc
//
