LOCUS       sequence-367291-        4672 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 4672)
  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 4672)
  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-348 pUC19-tNGFR-P2A-ICOS N
FEATURES             Location/Qualifiers
     source          1..4672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(42..64)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     164..183
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     377..399
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     391..408
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     392..408
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1757..1813
                     /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(2464..2480)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2464..2480)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2477..2499)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2488..2504
                     /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(2512..2518,2519..2536,2537..2542))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2557..2578
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2695..2712)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2866..3454)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2946..2965)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3625..4416,4417..4485))
                     /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     4248..4267
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4486..4590)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(4658..4672,1..4)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
       61 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
      121 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
      181 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
      241 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
      301 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
      361 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagctc
      421 ggtacccggg gataataatt ttgtttgttt cttgtcctat ttcaaaggct ttcttatctc
      481 tggagcacct agcataagat agaaatgtgt caaaatatat gttttattca tcatgtgagt
      541 atttttaggt cctgttaacc cccataacta ttgattcaga gaagtagggt ggttctgaaa
      601 aatacaggca taatctcttt aacttgtttt ataggaacca gaataagggt aatgttttcc
      661 tctgtcttca aaatcatcaa taatccatgc attgtttaac tcatgtcata agcaataatg
      721 cctttcatat agccattggc atcaaagaag aaacaccccc ttgatttgat ggtaagcgtg
      781 acactacata aactcccaga aaacccactt cctttccagc aaatagaaaa caaccgagag
      841 cctgaattca ctgtcagctt tgaacactga acgcgaggac tgttaactgt ttctggcaaa
      901 catgggggca ggtgccactg gccgcgccat ggacgggccg cgcctgctgc tgttgctgct
      961 tctgggggtg tcccttggag gtgccaagga ggcatgcccc acaggcctgt acacacacag
     1021 cggtgagtgc tgcaaagcct gcaacctggg cgagggtgtg gcccagcctt gtggagccaa
     1081 ccagaccgtg tgtgagccct gcctggacag cgtgacgttc tccgacgtgg tgagcgcgac
     1141 cgagccgtgc aagccgtgca ccgagtgcgt ggggctccag agcatgtcgg cgccatgcgt
     1201 ggaggccgac gacgccgtgt gccgctgcgc ctacggctac taccaggatg agacgactgg
     1261 gcgctgcgag gcgtgccgcg tgtgcgaggc gggctcgggc ctcgtgttct cctgccagga
     1321 caagcagaac accgtgtgcg aggagtgccc cgacggcacg tattccgacg aggccaacca
     1381 cgtggacccg tgcctgccct gcaccgtgtg cgaggacacc gagcgccagc tccgcgagtg
     1441 cacacgctgg gccgacgccg agtgcgagga gatccctggc cgttggatta cacggtccac
     1501 acccccagag ggctcggaca gcacagcccc cagcacccag gagcctgagg cacctccaga
     1561 acaagacctc atagccagca cggtggcagg tgtggtgacc acagtgatgg gcagctccca
     1621 gcccgtggtg acccgaggca ccaccgacaa cctcatccct gtctattgct ccatcctggc
     1681 tgctgtggtt gtgggtcttg tggcctacat agccttcaag aggtggaaca gccgcgccaa
     1741 gcgctcgggt tcgggtgcca ccaacttcag cctgctgaag caggccggcg acgtggagga
     1801 gaaccccggc cccatgaagt caggactctg gtatttcttt ctcttctgct tgcgcattaa
     1861 agttttaaca ggtaagtggt gtattgaata tttcttatta agttataatt caagtaaaca
     1921 ttaagaaaaa gcaaaggtag aaaaattacg cacccaaaag acagtggttt tggtttttga
     1981 aagtaggaat agtttcagga tatggccaag ggcaccatgt cactcatgtc actgtggatg
     2041 gcattgagtt tgcattgctg cctttgatac tgtgtcttga gatgaaaaac atgtttcatc
     2101 agaccattaa atttttaaat tacctgcttg gagttacctg tcagtggata aactagcaga
     2161 gtggcttact gttcttagag aaagatgaaa ggcaaaatga cttgtgtcaa cagaatgaaa
     2221 attaaatagc tctggcattt ggactgggaa ttctttctgt ggtagaaaat aaaatgggaa
     2281 gtctttgaaa agaaggattt tgattttaat cttacctgga tgatctgtta tctgaaatgt
     2341 ccagggcacc atagagtttt tattgacact aagtatcaga gttcacccta aaaagccaag
     2401 gaggaaagga taatgtaatc ctctagagtc gacctgcagg catgcaagct tggcgtaatc
     2461 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2521 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2581 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2641 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2701 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2761 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2821 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2881 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2941 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3001 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3061 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3121 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3181 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3241 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3301 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3361 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3421 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3481 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3541 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3601 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3661 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3721 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3781 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3841 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3901 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3961 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4021 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4081 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4141 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4201 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4261 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4321 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4381 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4441 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4501 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4561 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4621 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cg
//
