LOCUS       sequence-367332-        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-378 tNGFR-P2A-CCR7
FEATURES             Location/Qualifiers
     source          1..4398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..54)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     154..173
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     367..389
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     381..398
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     382..398
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1678..1734
                     /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(2180..2196)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2180..2196)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2193..2215)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2204..2220
                     /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(2228..2234,2235..2252,2253..2258))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2273..2294
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2411..2428)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2582..3170)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2662..2681)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3341..4132,4133..4201))
                     /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     3964..3983
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4202..4306)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4374..4392
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
       61 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
      121 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
      181 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
      241 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
      301 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      361 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagctc ggtacccggg
      421 gaacttctct cacacatctc tgactcccca acatctaacc catggcctgg catgtgatat
      481 tcatgctata aatatttgtt gaatcagtaa atggcccctc atctagattc tgccaggcag
      541 cgcagggggc tttttgaatg taaatgacct aatgattcag gatcctatga ccagcgactg
      601 tcaccctctg tcacctttct tctgcccctt tatctctgaa ggcattgaag gggccctgca
      661 tgagtcaggg agggctgggg ggaggagcaa gggtgggagg ggcagggaag agggtggctt
      721 ctccgacaac ttaaaagggg cttgaaccac ttcctcccca gacaggggta gtgcgaggcc
      781 gggcacagcc ttcctgtgtg gttttaccgc ccagagagcg tcatgggggc aggtgccact
      841 ggccgcgcca tggacgggcc gcgcctgctg ctgttgctgc ttctgggggt gtcccttgga
      901 ggtgccaagg aggcatgccc cacaggcctg tacacacaca gcggtgagtg ctgcaaagcc
      961 tgcaacctgg gcgagggtgt ggcccagcct tgtggagcca accagaccgt gtgtgagccc
     1021 tgcctggaca gcgtgacgtt ctccgacgtg gtgagcgcga ccgagccgtg caagccgtgc
     1081 accgagtgcg tggggctcca gagcatgtcg gcgccatgcg tggaggccga cgacgccgtg
     1141 tgccgctgcg cctacggcta ctaccaggat gagacgactg ggcgctgcga ggcgtgccgc
     1201 gtgtgcgagg cgggctcggg cctcgtgttc tcctgccagg acaagcagaa caccgtgtgc
     1261 gaggagtgcc ccgacggcac gtattccgac gaggccaacc acgtggaccc gtgcctgccc
     1321 tgcaccgtgt gcgaggacac cgagcgccag ctccgcgagt gcacacgctg ggccgacgcc
     1381 gagtgcgagg agatccctgg ccgttggatt acacggtcca cacccccaga gggctcggac
     1441 agcacagccc ccagcaccca ggagcctgag gcacctccag aacaagacct catagccagc
     1501 acggtggcag gtgtggtgac cacagtgatg ggcagctccc agcccgtggt gacccgaggc
     1561 accaccgaca acctcatccc tgtctattgc tccatcctgg ctgctgtggt tgtgggtctt
     1621 gtggcctaca tagccttcaa gaggtggaac agccgcgcca agcgctcggg ttcgggtgcc
     1681 accaacttca gcctgctgaa gcaggccggc gacgtggagg agaaccccgg ccccgacctg
     1741 ggtgagtgag cctcttcatg tgagaaggaa cagtaccagg gtcttggaca cccagactga
     1801 ccctgtggaa tgggggtgga ggatgcgggt ggagcgcata ggggtgcttc ctggagaaat
     1861 cagttctgat ttggtgggca ttggaagcta cttccagaga atgccccctg ctgtcccagg
     1921 aggggtgcac ctgccctttc agacctggag aaggggtcag cctttggtgc cagtggggac
     1981 aagggagggg ccgagatcat gtctttctaa aacagcaggg acataagaga gtggcggggc
     2041 tgggcttggt ggtctggcag aacctgttct catcatgagg gtgataggtt aggccctctg
     2101 ggacctctgg ggacaggttt caacagagaa attctcctct agagtcgacc tgcaggcatg
     2161 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2221 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     2281 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2341 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2401 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2461 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2521 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2581 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2641 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2701 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2761 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2821 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2881 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2941 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3001 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3061 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3121 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3181 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3241 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3301 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3361 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3421 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3481 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3541 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3601 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3661 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3721 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3781 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3841 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3901 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3961 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4021 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4081 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4141 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4201 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4261 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4321 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4381 gtatcacgag gccctttc
//
