LOCUS       sequence-367323-        4122 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 4122)
  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 4122)
  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-375 tNGFR-P2A-ITGB7
FEATURES             Location/Qualifiers
     source          1..4122
                     /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             1590..1646
                     /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(1904..1920)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1904..1920)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1917..1939)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1928..1944
                     /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(1952..1958,1959..1976,1977..1982))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1997..2018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2135..2152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2306..2894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2386..2405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3065..3856,3857..3925))
                     /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     3688..3707
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3926..4030)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4098..4116
                     /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 gaggtttcgc catgttgact aggctggttt cgaattcctg agctcaggtg atccacccac
      481 cttggcttcc taaagtgctg agataacagg catgagccac cacgtccgga ggcccctacc
      541 tttgcgtgat acctgctcct tcccattatt ttgaagaaac tgctgaatcc acaggggaag
      601 tgaaactatg aggggagata aagtgttggg gcagagtttt gagagtgata accacaagct
      661 aagtttagag gactgagagt ttgagagtcc tccctgagga cccacatgtc cccctttata
      721 tcctattaca gggcatgggg gcaggtgcca ctggccgcgc catggacggg ccgcgcctgc
      781 tgctgttgct gcttctgggg gtgtcccttg gaggtgccaa ggaggcatgc cccacaggcc
      841 tgtacacaca cagcggtgag tgctgcaaag cctgcaacct gggcgagggt gtggcccagc
      901 cttgtggagc caaccagacc gtgtgtgagc cctgcctgga cagcgtgacg ttctccgacg
      961 tggtgagcgc gaccgagccg tgcaagccgt gcaccgagtg cgtggggctc cagagcatgt
     1021 cggcgccatg cgtggaggcc gacgacgccg tgtgccgctg cgcctacggc tactaccagg
     1081 atgagacgac tgtgcgctgc gaggcgtgcc gcgtgtgcga ggcgggctcg ggcctcgtgt
     1141 tctcctgcca ggacaagcag aacaccgtgt gcgaggagtg ccccgacggc acgtattccg
     1201 acgaggccaa ccacgtggac ccgtgcctgc cctgcaccgt gtgcgaggac accgagcgcc
     1261 agctccgcga gtgcacacgc tgggccgacg ccgagtgcga ggagatccct ggccgttgga
     1321 ttacacggtc cacaccccca gagggctcgg acagcacagc ccccagcacc caggagcctg
     1381 aggcacctcc agaacaagac ctcatagcca gcacggtggc aggtgtggtg accacagtga
     1441 tgggcagctc ccagcccgtg gtgacccgag gcaccaccga caacctcatc cctgtctatt
     1501 gctccatcct ggctgctgtg gttgtgggtc ttgtggccta catagccttc aagaggtgga
     1561 acagccgcgc caagcgctcg ggttcgggtg ccaccaactt cagcctgctg aagcaggccg
     1621 gcgacgtgga ggagaacccc ggccccgtcg cattacctat ggtccttgtt ttgctgctgg
     1681 tcctgagcag aggtgagagt gaattggacg ccaagatccc atccacaggg gatgccacag
     1741 aatggcggaa tcctcacctg tccatgctgg ggtcctgcca gccagccccc tcctgccaga
     1801 agtgcatcct ctcacacccc agctgtgcat ggtgcaagca actggtaaag atgggccttc
     1861 ctctagagtc gacctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg
     1921 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     1981 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     2041 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2101 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2161 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2221 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2281 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2341 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2401 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2461 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2521 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2581 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2641 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2701 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2761 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2821 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2881 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2941 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3001 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3061 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3121 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3181 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3241 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3301 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3361 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3421 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3481 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3541 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3601 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3661 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3721 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3781 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3841 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3901 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3961 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4021 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     4081 tgacattaac ctataaaaat aggcgtatca cgaggccctt tc
//
