LOCUS       sequence-367272-        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-373 tNGFR-P2A-IL7R
FEATURES             Location/Qualifiers
     source          1..4398
                     /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             1680..1736
                     /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(2182..2198)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2182..2198)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2195..2217)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2206..2222
                     /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(2230..2236,2237..2254,2255..2260))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2275..2296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2413..2430)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2584..3172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2664..2683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3343..4134,4135..4203))
                     /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     3966..3985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4204..4308)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4376..4394
                     /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 gggaaagcaa gactctgtct caaaaaaata aaaattaaaa aaataaagta gcctctagcc
      481 taagatagct tgagcctagg tgtgaatcta ctgccttact ctgatgtaag cacagtaagt
      541 gtgggggctg cagggaatat ccaggaggaa caataatttc agaggctctg tctcttcatg
      601 tccttgacct ctgcttacag cagcaatact tttactcaga cttcctgttt ctggaacttg
      661 ccttcttttt tgctgtgttt atacttccct tgtctgtggt tagataagta taaagcccta
      721 gatctaagct tctctgtctt cctccctccc tcccttcctc ttactctcat tcatttcata
      781 cacactggct cacacatcta ctctctctct ctatctctct cagaatgggg gcaggtgcca
      841 ctggccgcgc catggacggg ccgcgcctgc tgctgttgct gcttctgggg gtgtcccttg
      901 gaggtgccaa ggaggcatgc cccacaggcc tgtacacaca cagcggtgag tgctgcaaag
      961 cctgcaacct gggcgagggt gtggcccagc cttgtggagc caaccagacc gtgtgtgagc
     1021 cctgcctgga cagcgtgacg ttctccgacg tggtgagcgc gaccgagccg tgcaagccgt
     1081 gcaccgagtg cgtggggctc cagagcatgt cggcgccatg cgtggaggcc gacgacgccg
     1141 tgtgccgctg cgcctacggc tactaccagg atgagacgac tgggcgctgc gaggcgtgcc
     1201 gcgtgtgcga ggcgggctcg ggcctcgtgt tctcctgcca ggacaagcag aacaccgtgt
     1261 gcgaggagtg ccccgacggc acgtattccg acgaggccaa ccacgtggac ccgtgcctgc
     1321 cctgcaccgt gtgcgaggac accgagcgcc agctccgcga gtgcacacgc tgggccgacg
     1381 ccgagtgcga ggagatccct ggccgttgga ttacacggtc cacaccccca gagggctcgg
     1441 acagcacagc ccccagcacc caggagcctg aggcacctcc agaacaagac ctcatagcca
     1501 gcacggtggc aggtgtggtg accacagtga tgggcagctc ccagcccgtg gtgacccgag
     1561 gcaccaccga caacctcatc cctgtctatt gctccatcct ggctgctgtg gttgtgggtc
     1621 ttgtggccta catagccttc aagaggtgga acagccgcgc caagcgctcg ggttcgggtg
     1681 ccaccaactt cagcctgctg aagcaggccg gcgacgtgga ggagaacccc ggccccacaa
     1741 ttctaggaac tacattcggg atggtttttt ctttacttca agtcgtttct ggagaaagtg
     1801 gctatgctca aaatggtgag tcatttctaa gttttcttat ggattttgga ttatctgtag
     1861 catggtttca ggttattcag ttccctaaca gacctgagtc aggcactggg tttgaatgca
     1921 gtttgagaat ttcccacata ttcagtcatt ttttttaatg tttaaccacc atgacagggg
     1981 gcaggggatc aatactatgg gtggtttata agacctcagt attctcaaga aggaatgcat
     2041 ttcactccca agtgtagatc ttaaatgttg aatgattact ctgctcttac aaaaagaatg
     2101 ctcatgtaga tgctatgact gtacttgtag gaaaattcct ctagagtcga cctgcaggca
     2161 tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2221 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     2281 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2341 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2401 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2461 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2521 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2581 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2641 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2701 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2761 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2821 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2881 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2941 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3001 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3061 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3121 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3181 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3241 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3301 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3361 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3421 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3481 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3541 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3601 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3661 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3721 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3781 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3841 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3901 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3961 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4021 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4081 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4141 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4201 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4261 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4321 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     4381 gcgtatcacg aggccctt
//
