LOCUS       sequence-367285-        4060 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 4060)
  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 4060)
  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-472 FOXO1-tNGFR
FEATURES             Location/Qualifiers
     source          1..4060
                     /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             1493..1549
                     /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(1843..1859)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1843..1859)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1856..1878)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1867..1883
                     /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(1891..1897,1898..1915,1916..1921))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1936..1957
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2074..2091)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2245..2833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2325..2344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3004..3795,3796..3864))
                     /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     3627..3646
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3865..3969)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4037..4055
                     /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 ggacgaggag cctcgatgtg gatggccccg cgaagttaag ttctgggctc gcgcttccac
      481 tccgccgcgc cttcctccca gtttccgtcc gctcgccgca ccggcttcgt tcccccaaat
      541 ctcggaccgt cccttcgcgc cccctccccg tccgccccca gtgctgcgtt ctccccctct
      601 tggctctcct gcggctgggg gaggggcggg ggtcaccatg ggggcaggtg ccactggccg
      661 cgccatggac gggccgcgcc tgctgctgtt gctgcttctg ggggtgtccc ttggaggtgc
      721 caaggaggca tgccccacag gcctgtacac acacagcggt gagtgctgca aagcctgcaa
      781 cctgggcgag ggtgtggccc agccttgtgg agccaaccag accgtgtgtg agccctgcct
      841 ggacagcgtg acgttctccg acgtggtgag cgcgaccgag ccgtgcaagc cgtgcaccga
      901 gtgcgtgggg ctccagagca tgtcggcgcc atgcgtggag gccgacgacg ccgtgtgccg
      961 ctgcgcctac ggctactacc aggatgagac gactgggcgc tgcgaggcgt gccgcgtgtg
     1021 cgaggcgggc tcgggcctcg tgttctcctg ccaggacaag cagaacaccg tgtgcgagga
     1081 gtgccccgac ggcacgtatt ccgacgaggc caaccacgtg gacccgtgcc tgccctgcac
     1141 cgtgtgcgag gacaccgagc gccagctccg cgagtgcaca cgctgggccg acgccgagtg
     1201 cgaggagatc cctggccgtt ggattacacg gtccacaccc ccagagggct cggacagcac
     1261 agcccccagc acccaggagc ctgaggcacc tccagaacaa gacctcatag ccagcacggt
     1321 ggcaggtgtg gtgaccacag tgatgggcag ctcccagccc gtggtgaccc gaggcaccac
     1381 cgacaacctc atccctgtct attgctccat cctggctgct gtggttgtgg gtcttgtggc
     1441 ctacatagcc ttcaagaggt ggaacagccg cgccaagcgc tcgggttcgg gtgccaccaa
     1501 cttcagcctg ctgaagcagg ccggcgacgt ggaggagaac cctggcccta tggccgaggc
     1561 gcctcaggtg gtggagatcg acccggactt cgagccgctg ccccggccgc gctcgtgcac
     1621 ctggccgctg cccaggccgg agtttagcca gtccaactcg gccacctcca gcccggcgcc
     1681 gtcgggcagc gcggctgcca accccgacgc cgcggcgggc ctgccctcgg cctcggctgc
     1741 cgctgtcagc gccgacttca tgagcaacct gagcttgctg gaggagagcg aggactttcc
     1801 tctagagtcg acctgcaggc atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt
     1861 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     1921 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     1981 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2041 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2101 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatatggt tatccacaga
     2161 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2221 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2281 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2341 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2401 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2461 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2521 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2581 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2641 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     2701 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     2761 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     2821 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     2881 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     2941 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3001 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3061 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3121 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3181 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3241 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3301 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3361 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3421 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3481 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3541 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3601 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3661 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3721 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3781 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3841 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     3901 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3961 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     4021 gacattaacc tataaaaata ggcgtatcac gaggcccttt
//
