LOCUS       sequence-367290-        4274 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 4274)
  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 4274)
  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-346 pUC19-tNGFR-P2A-IL2RA N
FEATURES             Location/Qualifiers
     source          1..4274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(34..56)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     156..175
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     369..391
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     383..400
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     384..400
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1538..1594
                     /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(2058..2074)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2058..2074)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2071..2093)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2082..2098
                     /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(2106..2112,2113..2130,2131..2136))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2151..2172
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2289..2306)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2460..3048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2540..2559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3219..4010,4011..4079))
                     /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     3842..3861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4080..4184)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4252..4270
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
       61 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
      121 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
      181 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
      241 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
      301 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
      361 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcgagc tcggtacccg
      421 gggaaaacac ttcctatatt tgagatgaga gaagagagtg ctaggcagtt tcctggctga
      481 acacgccagc ccaatactta aagagagcaa ctcctgactc cgatagagac tggatggacc
      541 cacaagggtg acagcccagg cggaccgatc ttcccatccc acatcctccg gcgcgatgcc
      601 aaaaagaggc tgacggcaac tgggccttct gcagagaaag acctccgctt cactgccccg
      661 gctggtccca agggtcagga agatgggggc aggtgccact ggccgcgcca tggacgggcc
      721 gcgcctgctg ctgttgctgc ttctgggggt gtcccttgga ggtgccaagg aggcatgccc
      781 cacaggcctg tacacacaca gcggtgagtg ctgcaaagcc tgcaacctgg gcgagggtgt
      841 ggcccagcct tgtggagcca accagaccgt gtgtgagccc tgcctggaca gcgtgacgtt
      901 ctccgacgtg gtgagcgcga ccgagccgtg caagccgtgc accgagtgcg tggggctcca
      961 gagcatgtcg gcgccatgcg tggaggccga cgacgccgtg tgccgctgcg cctacggcta
     1021 ctaccaggat gagacgactg ggcgctgcga ggcgtgccgc gtgtgcgagg cgggctcggg
     1081 cctcgtgttc tcctgccagg acaagcagaa caccgtgtgc gaggagtgcc ccgacggcac
     1141 gtattccgac gaggccaacc acgtggaccc gtgcctgccc tgcaccgtgt gcgaggacac
     1201 cgagcgccag ctccgcgagt gcacacgctg ggccgacgcc gagtgcgagg agatccctgg
     1261 ccgttggatt acacggtcca cacccccaga gggctcggac agcacagccc ccagcaccca
     1321 ggagcctgag gcacctccag aacaagacct catagccagc acggtggcag gtgtggtgac
     1381 cacagtgatg ggcagctccc agcccgtggt gacccgaggc accaccgaca acctcatccc
     1441 tgtctattgc tccatcctgg ctgctgtggt tgtgggtctt gtggcctaca tagccttcaa
     1501 gaggtggaac agccgcgcca agcgctcggg ttcgggtgcc accaacttca gcctgctgaa
     1561 gcaggccggc gacgtggagg agaaccccgg ccccatggat tcatatctgc tgatgtgggg
     1621 actgctcacg ttcatcatgg tgcctggctg ccaggcaggt aagggcctgt gggtgccccc
     1681 ggaattccgg gaaggctgat gggcatccct cttcccagcc acagaaccag agggagtccc
     1741 caggtagatg gttccaagaa gggagttgaa tcttgggttc cacctcttgc ctgtgaccca
     1801 cggggacccc agtttatgcc tcactgttcc ttggtctgtc aagagagcct gaaatagcat
     1861 taggttctcc tgtccttctc agtccttgac aattaattct gggaagaata gtgtggcatg
     1921 atatttggga tatttggatg ttaacagggt cccgatgagc aggtttctca agaaattatc
     1981 ttttattctt caatgaattt ccttttattc tctcctctag agtcgacctg caggcatgca
     2041 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2101 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2161 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2221 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2281 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2341 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2401 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2461 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2521 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2581 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2641 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2701 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2761 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2821 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2881 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2941 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3001 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3061 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3121 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3181 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3241 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3301 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3361 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3421 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3481 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3541 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3601 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3661 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3721 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3781 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3841 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3901 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3961 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4021 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4081 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4141 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4201 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     4261 atcacgaggc cctt
//
