LOCUS       sequence-367439-        4388 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 4388)
  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 4388)
  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-339 pUC19-tNGFR-P2A-TIM3 N
FEATURES             Location/Qualifiers
     source          1..4388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(36..58)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     158..177
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     371..393
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     385..402
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     386..402
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1661..1717
                     /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(2174..2190)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2174..2190)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2187..2209)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2198..2214
                     /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(2222..2228,2229..2246,2247..2252))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2267..2288
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2405..2422)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2576..3164)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2656..2675)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3335..4126,4127..4195))
                     /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     3958..3977
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4196..4300)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4368..4386
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
       61 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
      121 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
      181 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
      241 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
      301 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
      361 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaaatcga gctcggtacc
      421 cggggagctc catttcaggt gtatctcatc tgagacatta ttagagggtt ctctttatgt
      481 gccatgactg agtagcgttt tcttccactg aagtattgct tcctctttgt tgatatcaca
      541 ggacagacat cagaacactt acaggatgtg tgtagtgtgg catgacagag aactttggtt
      601 tcctttaatg tgactgtaga cctggcagtg ttactataag aatcactggc aatcagacac
      661 ccgggtgtgc tgagctagca ctcagtgggg gcggctactg ctcatgtgat tgtggagtag
      721 acagttggaa gaagtaccca gtccatttgg agagttaaaa ctgtgcctaa cagaggtgtc
      781 ctctgacttt tcttctgcaa gctccatggg ggcaggtgcc actggccgcg ccatggacgg
      841 gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca aggaggcatg
      901 ccccacaggc ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg
      961 tgtggcccag ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac
     1021 gttctccgac gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct
     1081 ccagagcatg tcggcgccat gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg
     1141 ctactaccag gatgagacga ctgggcgctg cgaggcgtgc cgcgtgtgcg aggcgggctc
     1201 gggcctcgtg ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg
     1261 cacgtattcc gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga
     1321 caccgagcgc cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc
     1381 tggccgttgg attacacggt ccacaccccc agagggctcg gacagcacag cccccagcac
     1441 ccaggagcct gaggcacctc cagaacaaga cctcatagcc agcacggtgg caggtgtggt
     1501 gaccacagtg atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat
     1561 ccctgtctat tgctccatcc tggctgctgt ggttgtgggt cttgtggcct acatagcctt
     1621 caagaggtgg aacagccgcg ccaagcgctc gggttcgggt gccaccaact tcagcctgct
     1681 gaagcaggcc ggcgacgtgg aggagaaccc cggccccttt tcacatcttc cgtttgactg
     1741 tgtcctgctg ctgctgctgc tactacttac aagtaagtct cggcatggat atttacaatg
     1801 acataatggt gctgtacaga ggacaaggga gagatacaag gatggccatt gtaatgttgt
     1861 ttgtaatagc aaaagcttgg aagcaaccta aatgtccgtc gatgagggag tgggtaaata
     1921 aatgtgatat aatcatgata caaacagtga gtctgacaaa acatactggt atagaatgtc
     1981 tcaaaattat aatgttatta aagaaaaaaa gttgcagaat aatgcatata ttaggcttct
     2041 atttgtgtgt gtgcattgtc ggggagcagg aatatgtgga catttcttaa tgtataagct
     2101 ctaggaaaag gaaccaggga atggtaaatc ctctagagtc gacctgcagg catgcaagct
     2161 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2221 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2281 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2341 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2401 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2461 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2521 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2581 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2641 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2701 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2761 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2821 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2881 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2941 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3001 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3061 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3121 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3181 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3241 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3301 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3361 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3421 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3481 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3541 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3601 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3661 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3721 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3781 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3841 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3901 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3961 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4021 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4081 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4141 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4201 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4261 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4321 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4381 cgaggccc
//
