LOCUS       sequence-367287-        4295 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 4295)
  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 4295)
  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-345 pUC19-tNGFR-P2A-IL2RB N
FEATURES             Location/Qualifiers
     source          1..4295
                     /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             1715..1771
                     /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(2078..2094)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2078..2094)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2091..2113)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2102..2118
                     /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(2126..2132,2133..2150,2151..2156))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2171..2192
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2309..2326)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2480..3068)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2560..2579)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3239..4030,4031..4099))
                     /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     3862..3881
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4100..4204)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4272..4290
                     /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 ggacttgccc ggctaatttt tgtattttta gtagagacag ggtttcacca tgttggtcag
      481 gctggtctca aactcttgac ctcaggtgat tcgtctgcct cgacctccca aagtgctggg
      541 attacaggca tgagccacac gcctggccac gacgggtgct tctttatgta gtctagtcaa
      601 agaagacctc tcaaaggagg ctgcagttga gcagaaactt gaaggaaggg tgtgagcatg
      661 tgtagggtgg tgggattagc aggtgcaaag gccctggggc tggactgagc tggcaccgag
      721 gagtgcagac catgcacact gagggggcgg gcccagcctg cctctagggg cagcgaggcc
      781 tgggatgtgt atttacgtgt gctctctctt tctcctctcc caccagggct tccttcctcg
      841 gctccaccct gtggatgtaa tgggggcagg tgccactggc cgcgccatgg acgggccgcg
      901 cctgctgctg ttgctgcttc tgggggtgtc ccttggaggt gccaaggagg catgccccac
      961 aggcctgtac acacacagcg gtgagtgctg caaagcctgc aacctgggcg agggtgtggc
     1021 ccagccttgt ggagccaacc agaccgtgtg tgagccctgc ctggacagcg tgacgttctc
     1081 cgacgtggtg agcgcgaccg agccgtgcaa gccgtgcacc gagtgcgtgg ggctccagag
     1141 catgtcggcg ccatgcgtgg aggccgacga cgccgtgtgc cgctgcgcct acggctacta
     1201 ccaggatgag acgactgggc gctgcgaggc gtgccgcgtg tgcgaggcgg gctcgggcct
     1261 cgtgttctcc tgccaggaca agcagaacac cgtgtgcgag gagtgccccg acggcacgta
     1321 ttccgacgag gccaaccacg tggacccgtg cctgccctgc accgtgtgcg aggacaccga
     1381 gcgccagctc cgcgagtgca cacgctgggc cgacgccgag tgcgaggaga tccctggccg
     1441 ttggattaca cggtccacac ccccagaggg ctcggacagc acagccccca gcacccagga
     1501 gcctgaggca cctccagaac aagacctcat agccagcacg gtggcaggtg tggtgaccac
     1561 agtgatgggc agctcccagc ccgtggtgac ccgaggcacc accgacaacc tcatccctgt
     1621 ctattgctcc atcctggctg ctgtggttgt gggtcttgtg gcctacatag ccttcaagag
     1681 gtggaacagc cgcgccaagc gctcgggttc gggtgccacc aacttcagcc tgctgaagca
     1741 ggccggcgac gtggaggaga accccggccc catggcagca cctgctctgt cctggcgtct
     1801 gcccctcctc atcctcctcc tgcccctggc tacctcttgg gcatctgcag cggtgaatgg
     1861 tgaggaccct gacatctgaa cagctctgct ttccctatgg agagatggct aaggccctgc
     1921 taaagcctct tcccagaagg ggatggggcc agagaccagg tgtccacctc ctgcctcacc
     1981 ctctgtcctg gacccctgcc ctggcatgca tgcatgaatg cgcgcacaca catcctctag
     2041 agtcgacctg caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2101 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2161 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2221 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2281 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2341 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2401 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2461 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2521 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2581 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2641 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2701 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2761 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2821 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2881 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2941 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3001 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3061 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3121 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3181 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3241 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3301 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3361 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3421 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3481 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3541 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3601 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3661 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3721 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3781 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3841 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3901 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3961 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4021 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4081 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4141 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4201 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     4261 taacctataa aaataggcgt atcacgaggc ccttt
//
