LOCUS       sequence-367326-        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-377 tNGFR-P2A-CD5
FEATURES             Location/Qualifiers
     source          1..4398
                     /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(2185..2201)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2185..2201)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2198..2220)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2209..2225
                     /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(2233..2239,2240..2257,2258..2263))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2278..2299
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2416..2433)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2587..3175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2667..2686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3346..4137,4138..4206))
                     /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     3969..3988
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4207..4311)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4379..4397
                     /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 ccggggaacg cctgtcctca gcccagccct cagctgcagc caggccttca gcctccgtaa
      481 cccccgctca gggtccccac cccctgcagc cctgtccctc caggatgcat ggccttgtcc
      541 tgtgtggggg tggccgagag cactgcccca gccctgggta ccttgggcag gaagctggca
      601 gaggccaggg ctgccattca aacaggggca ggtggttttg ccaggaggaa gttgacagtt
      661 caacttcaaa catgggtgac gcaggcccca cactgcctgc tccccgtccc acccctccct
      721 gagcacgcca ccccgccctc tccctctctg agagcgagat acccggccag acaccctcac
      781 ctgcggtgcc cagctgccca ggctgaggca agagaaggcc agaaacaatg 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 gtgcgtgggg 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 cccggccccc
     1741 ccatggggtc tctgcaaccg ctggccacct tgtacctgct ggggatgctg ggtgagtacc
     1801 cctcccaggt gtcctgcgaa cacccgggct cgctccagtg caaggaagga gttcccagtt
     1861 ttacccaagg ctgactctgg gatccacatg tcagccctct ggagcgttgt ggagatttgg
     1921 ggccactggg atccctgcct gcccccacta agccgcagct tggccctctg tcctgcatgt
     1981 cccacccgcc aggagcacaa ccttgcctct ctcatgcgct gttgagaacc ctgctttacc
     2041 cttccagtgc aagagagact gcagggggga cccgcatttg atggggccca gacaacttga
     2101 ttcctaggct gagttggatt ttagcagagc attcaggcct cctctagagt cgacctgcag
     2161 gcatgcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     2221 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     2281 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2341 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2401 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2461 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2521 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2581 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2641 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2701 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2761 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2821 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2881 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2941 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3001 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3061 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3121 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3181 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3241 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3301 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3361 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3421 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3481 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3541 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3601 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3661 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3721 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3781 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3841 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3901 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3961 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4021 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4081 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4141 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4201 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4261 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4321 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     4381 taggcgtatc acgaggcc
//
