LOCUS       sequence-367430-        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-460 tNGFR-P2A-STAT4
FEATURES             Location/Qualifiers
     source          1..4398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..55)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     155..174
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     368..390
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     382..399
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     383..399
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1679..1735
                     /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(2181..2197)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2181..2197)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2194..2216)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2205..2221
                     /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(2229..2235,2236..2253,2254..2259))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2274..2295
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2412..2429)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2583..3171)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2663..2682)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3342..4133,4134..4202))
                     /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     3965..3984
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4203..4307)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4375..4393
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
       61 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
      121 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
      181 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
      241 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
      301 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
      361 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattcgagct cggtacccgg
      421 ggaggaggta tagtaaatgt caaatacgtt tgccttcaag ttgctccaag tctaaatgtt
      481 gaggggacaa gagataggga aagagcttgt tgataataga aattcacata gacagctgtg
      541 ggcaggatgt ggggtgtttg gcagagtggg agtgaaaact ttttggagca ggtagaacag
      601 gagttcattt tagagcaagg aaggctgtag tttaggggca cagaaagaag gctgctccct
      661 tcattagggt tgaattatga aaaaaaacct tagtgaaatt tttgtatcct tggatatatt
      721 ggaattcagt cataacattg actaattcta aggaaagaag aaaatatgta ctagggctat
      781 gaacaatatt ttaaaactct aattctacac cttcttttat agcatggggg caggtgccac
      841 tggccgcgcc atggacgggc cgcgcctgct gctgttgctg cttctggggg tgtcccttgg
      901 aggtgccaag gaggcatgcc ccacaggcct gtacacacac agcggtgagt gctgcaaagc
      961 ctgcaacctg ggcgagggtg tggcccagcc ttgtggagcc aaccagaccg tgtgtgagcc
     1021 ctgcctggac agcgtgacgt tctccgacgt ggtgagcgcg accgagccgt gcaagccgtg
     1081 caccgagtgc gtggggctcc agagcatgtc ggcgccatgc gtggaggccg acgacgccgt
     1141 gtgccgctgc gcctacggct actaccagga tgagacgact gggcgctgcg aggcgtgccg
     1201 cgtgtgcgag gcgggctcgg gcctcgtgtt ctcctgccag gacaagcaga acaccgtgtg
     1261 cgaggagtgc cccgacggca cgtattccga cgaggccaac cacgtggacc cgtgcctgcc
     1321 ctgcaccgtg tgcgaggaca ccgagcgcca gctccgcgag tgcacacgct gggccgacgc
     1381 cgagtgcgag gaaatccctg gccgttggat tacacggtcc acacccccag agggctcgga
     1441 cagcacagcc cccagcaccc aggagcctga ggcacctcca gaacaagacc tcatagccag
     1501 cacggtggca ggtgtggtga ccacagtgat gggcagctcc cagcccgtgg tgacccgagg
     1561 caccaccgac aacctcatcc ctgtctattg ctccatcctg gctgctgtgg ttgtgggtct
     1621 tgtggcctac atagccttca agaggtggaa cagccgcgcc aagcgctcgg gttcgggtgc
     1681 caccaacttc agcctgctga agcaggccgg cgacgtggag gagaaccccg gcccctctca
     1741 gtggaatcaa gtgcaacagt tagaaatcaa gtttttggag caggtggatc aattctatga
     1801 tgacaacttt cccatggaaa ttcggcatct gttggcccaa tggattgaaa atcaagactg
     1861 gtaggatcaa acatattttc cctagaagtt gatgcacaaa tgtctgatgc tctatccatg
     1921 tgaatttatt ttatggtcca ctttttactc agtagatgca ttcttttcag gtaaagaact
     1981 ttctcaagga tttgaaagcc ttcccaaaga aggggaataa ttgtcctttc tggttccatt
     2041 cattgtaaat gaaaagttaa tggttccagt gcttcttttc tctgtaaaca aaaacccaaa
     2101 taatttttca tgtattaaaa aaagaagcaa atcaatcctc tagagtcgac ctgcagacat
     2161 gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     2221 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     2281 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2341 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2401 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2461 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2521 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2581 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2641 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2701 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2761 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2821 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2881 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2941 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3001 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3061 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3121 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3181 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3241 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3301 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3361 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3421 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3481 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3541 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3601 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3661 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3721 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3781 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3841 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3901 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3961 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4021 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4081 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4141 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4201 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4261 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4321 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     4381 cgtatcacga ggcccttt
//
