LOCUS       sequence-367445-        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-452 tNGFR-P2A-Lck
FEATURES             Location/Qualifiers
     source          1..4398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..42)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     142..161
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     355..377
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     369..386
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     370..386
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1666..1722
                     /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(2168..2184)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2168..2184)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2181..2203)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2192..2208
                     /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(2216..2222,2223..2240,2241..2246))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2261..2282
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2399..2416)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2570..3158)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2650..2669)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3329..4120,4121..4189))
                     /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     3952..3971
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4190..4294)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4362..4380
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
       61 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
      121 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
      181 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat
      241 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
      301 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
      361 cacgacgttg taaaacgacg gccagtgaat tcgagctcgg tacccgggga aggtctctcc
      421 caggcttggg aaagtgtgtg tcatctctag gaggtggtcc tcccaacaca gggtactggc
      481 agagggagag ggagggggca gaggcaggaa gtgggtaact agactaacaa aggtgcctgt
      541 ggcggtttgc ccatcccagg tgggagggtg gggctagggc tcaggggccg tgtgtgaatt
      601 tacttgtagc ctgagggctc agagggagca ccggtttgga gctgggaccc cctattttag
      661 cttttctgtg gctggtgaat ggggatccca ggatctcaca atctcaggta cttttggaac
      721 tttccagggc aaggccccat tatatctgat gttgggggag cagatcttgg gggagcccct
      781 tcagccccct cttccattcc ctcagggacc atgggggcag gtgccactgg ccgcgccatg
      841 gacgggccgc gcctgctgct gttgctgctt ctgggggtgt cccttggagg tgccaaggag
      901 gcatgcccca caggcctgta cacacacagc ggtgagtgct gcaaagcctg caacctgggc
      961 gagggtgtgg cccagccttg tggagccaac cagaccgtgt gtgagccctg cctggacagc
     1021 gtgacgttct ccgacgtggt gagcgcgacc gagccgtgca agccgtgcac cgagtgcgtg
     1081 gggctccaga gcatgtcggc gccatgcgtg gaggccgacg acgccgtgtg ccgctgcgcc
     1141 tacggctact accaggatga gacgactggg cgctgcgagg cgtgccgcgt gtgcgaggcg
     1201 ggctcgggcc tcgtgttctc ctgccaggac aagcagaaca ccgtgtgcga ggagtgcccc
     1261 gacggcacgt attccgacga ggccaaccac gtggacccgt gcctgccctg caccgtgtgc
     1321 gaggacaccg agcgccagct ccgcgagtgc acacgctggg ccgacgccga gtgcgaggag
     1381 atccctggcc gttggattac acggtccaca cccccagagg gctcggacag cacagccccc
     1441 agcacccagg agcctgaggc acctccagaa caagacctca tagccagcac ggtggcaggt
     1501 gtggtgacca cagtgatggg cagctcccag cccgtggtga cccgaggcac caccgacaac
     1561 ctcatccctg tctattgctc catcctggct gctgtggttg tgggtcttgt ggcctacata
     1621 gccttcaaga ggtggaacag ccgcgccaag cgctcgggtt cgggtgccac caacttcagc
     1681 ctgctgaagc aggccggcga cgtggaggag aaccccggcc ccggctgtgg ctgcagctca
     1741 cacccggaag atgactggat ggaaaacatc gatgtgtgtg agaactgcca ttatcccata
     1801 gtcccactgg atggcaaggg cacggtaaga ggcgagacag gggccttggt gagggagttg
     1861 ggtagagaat gcaacccagg agaaagaaat gaccagcact acaggccctt gaaagaatag
     1921 agtggccctc tcccctgaaa tacagaaagg aaaagaggcc cagagagggg aagggaatct
     1981 cctaagatca cacagaaagt agttggtaaa ctcagggata acatctaacc aggctggaga
     2041 ggctgagagc agagcagggg ggaagggggc cagggtctga cccaatcttc tgctttctga
     2101 ccccaccctc atcccccact cctcctctag agtcgacctg caggcatgca agcttggcgt
     2161 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2221 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2281 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2341 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2401 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2461 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2521 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2581 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2641 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2701 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2761 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2821 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2881 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2941 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3001 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3061 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3121 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3181 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3241 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3301 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3361 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3421 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3481 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3541 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3601 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3661 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3721 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3781 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3841 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3901 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3961 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4021 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4081 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4141 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4201 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4261 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4321 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4381 cctttcgtct cgcgcgtt
//
