LOCUS       sequence-367273-        4400 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 4400)
  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 4400)
  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-457 tNGFR-P2A-STAT1
FEATURES             Location/Qualifiers
     source          1..4400
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..62)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     162..181
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     375..397
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     389..406
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     390..406
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1686..1742
                     /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(2190..2206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2190..2206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2203..2225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2214..2230
                     /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(2238..2244,2245..2262,2263..2268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2283..2304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2421..2438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2592..3180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2672..2691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3351..4142,4143..4211))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTIPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3974..3993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4212..4316)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(4384..4400,1..2)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
       61 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
      121 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
      181 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
      241 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
      301 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
      361 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tcgagctcgg
      421 tacccgggga gtggtgaaaa cagacaataa tgaaaataaa atatgtggta ggtcagatgg
      481 tggtgtaagt actatgaaga aaaataaacc agggaaaaga gagagcatgt ttccagggtg
      541 ggaggtggtt gcagtttaaa taaaagaggc cagagaaggc tgccctgata tgttgacaat
      601 aagcaaagac atgaagatgt gtttcgtgga ggagaaaaaa agttcatccc caaactcaat
      661 taaaaacagg ctttagacta taaatgccat ttaaataaaa tattcctggg acaaactatg
      721 ttttggatta agcaagatat ttaagagtac aacctgatgc taggaaggct ttctttggag
      781 ctatggtttc catatacata gaaagaatgt atattctttt tccctatagg atgggggcag
      841 gtgccactgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt ctgggggtgt
      901 cccttggagg tgccaaggag gcatgcccca caggcctgta cacacacagc ggtgagtgct
      961 gcaaagcctg caacctgggc gagggtgtgg cccagccttg tggagccaac cagaccgtgt
     1021 gtgagccctg cctggacagc gtgacgttct ccgacgtggt gagcgcgacc gagccgtgca
     1081 agccgtgcac cgagtgcgtg gggctccaga gcatgtcggc gccatgcgtg gaggccgacg
     1141 acgccgtgtg ccgctgcgcc tacggctact accaggatga gacgactggg cgctgcgagg
     1201 cgtgccgcgt gtgcgaggcg ggctcgggcc tcgtgttctc ctgccaggac aagcagaaca
     1261 ccgtgtgcga ggagtgcccc gacggcacgt attccgacga ggccaaccac gtggacccgt
     1321 gcctgccctg caccgtgtgc gaggacaccg agcgccagct ccgcgagtgc acacgctggg
     1381 ccgacgccga gtgcgaggag atccctggcc gttggattac acggtccaca cccccagagg
     1441 gctcggacag cacagccccc agcacccagg agcctgaggc acctccagaa caagacctca
     1501 tagccagcac ggtggcaggt gtggtgacca cagtgatggg cagctcccag cccgtggtga
     1561 cccgaggcac caccgacaac ctcatccctg tctattgctc catcctggct gctgtggttg
     1621 tgggtcttgt ggcctacata gccttcaaga ggtggaacag ccgcgccaag cgctcgggtt
     1681 cgggtgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag aaccccggcc
     1741 cctctcagtg gtacgaactt cagcagcttg actcaaaatt cctggagcag gttcaccagc
     1801 tttatgatga cagttttccc atggaaatca gacagtacct ggcacagtgg ttagaaaagc
     1861 aagactggta aggaaaattc actagacaga agaagaagtt tacaccttaa aagtactggt
     1921 tgattaagtg acttggggcc atgtttactg gaagatgaat tccatcaatg gccactggaa
     1981 ttatttgttg tagaaaagtt tgttgattta aaaaatgact caacaaagtt gagccaaaaa
     2041 gtgtctcaac aaagttaagt atattagtca ttaagcactt ttatacagtt ttgccaaatt
     2101 taatttatga tagatattcc taaatgcttt ggacaagtta cggatcctct agagtcgacc
     2161 tgcaggcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2221 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2281 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2341 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2401 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2461 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2521 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2581 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2641 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2701 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2761 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2821 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2881 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2941 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3001 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3061 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3121 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3181 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3241 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3301 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3361 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3421 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3481 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3541 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3601 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3661 attgctacag gtatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3721 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3781 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3841 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3901 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3961 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4021 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4081 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4141 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4201 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4261 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4321 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     4381 aaaaataggc gtatcacgag
//
