LOCUS       sequence-367292-        4705 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 4705)
  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 4705)
  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-340 pUC19-tNGFR-P2A-IL2RG N
FEATURES             Location/Qualifiers
     source          1..4705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1816..1872
                     /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(2484..2500)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2484..2500)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2497..2519)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2508..2524
                     /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(2532..2538,2539..2556,2557..2562))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2577..2598
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2715..2732)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2886..3474)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2966..2985)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3645..4436,4437..4505))
                     /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     4268..4287
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4506..4610)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4678..4696
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggac
      421 agctaagggt gggtattctg gtttggatta gatcagagga aagacagctg tatatgtgcc
      481 cacaggagcc aagacggtat tttccatcct cccaaaacag tagagctttg acagagattt
      541 aagggtgacc aagtcaagga agaggcatgg catagaacgg tgatgtcggg ggtgggggtt
      601 cagaacttcc attatagaag gtaatgattt agaggagaag gtggttgaga atggtgctag
      661 tggtagtgaa cagatccttc ccaggatcta ggtgggctga ggatttttga gtctgtgaca
      721 ctattgtata tccagcttta gtttctgttt accaccttac agcagcacct aatctcctag
      781 aggacttagc ccgtgtcaca cagcacatat ttgccacacc ctctgtaaag ccctggttta
      841 taaggttctt tccaccggaa gctatgacag aggaaacgtg tgggtgggga ggggtagtgg
      901 gtgagggacc caggttcctg acacagacag actacaccca gggaatgaag agcaagcgcc
      961 atgggggcag gtgccactgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt
     1021 ctgggggtgt cccttggagg tgccaaggag gcatgcccca caggcctgta cacacacagc
     1081 ggtgagtgct gcaaagcctg caacctgggc gagggtgtgg cccagccttg tggagccaac
     1141 cagaccgtgt gtgagccctg cctggacagc gtgacgttct ccgacgtggt gagcgcgacc
     1201 gagccgtgca agccgtgcac cgagtgcgtg gggctccaga gcatgtcggc gccatgcgtg
     1261 gaggccgacg acgccgtgtg ccgctgcgcc tacggctact accaggatga gacgactggg
     1321 cgctgcgagg cgtgccgcgt gtgcgaggcg ggctcgggcc tcgtgttctc ctgccaggac
     1381 aagcagaaca ccgtgtgcga ggagtgcccc gacggcacgt attccgacga ggccaaccac
     1441 gtggacccgt gcctgccctg caccgtgtgc gaggacaccg agcgccagct ccgcgagtgc
     1501 acacgctggg ccgacgccga gtgcgaggag atccctggcc gttggattac acggtccaca
     1561 cccccagagg gctcggacag cacagccccc agcacccagg agcctgaggc acctccagaa
     1621 caagacctca tagccagcac ggtggcaggt gtggtgacca cagtgatggg cagctcccag
     1681 cccgtggtga cccgaggcac caccgacaac ctcatccctg tctattgctc catcctggct
     1741 gctgtggttg tgggtcttgt ggcctacata gccttcaaga ggtggaacag ccgcgccaag
     1801 cgctcgggtt cgggtgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag
     1861 aaccccggcc ccatgttgaa gccatcgttg ccgtttacct ccctcttatt cctgcagctg
     1921 cccctgctgg gagtggggct gaacacgaca attctgacgc ccaatgggaa tgaagacacc
     1981 acagctggtg ggaaatctgg gactggaggg ggctggtgag aagggtggct gtgggaaggg
     2041 gccgtacaga gatctggtgc ctgccactgg ccattacaat catgtgggca gaattgaaaa
     2101 gtggagtggg aagggcaagg gggagggttc cctgcctcac gctacttctt ctttctttct
     2161 tgtttgtttg tttctttctt tcttttgagg cagggtctca ctatgttgcc taggctggtc
     2221 tcaaactcct ggcctctagt gatcctcctg cctcagcctt tcaaagcacc aggattacag
     2281 acatgagcca ccgtgcttgg cctcctcctt ctgaccatca tttctctttc cctccctgcc
     2341 ttcattttct ccccaatcta gatttcttcc tgaccactat gcccactgac tccctcagtg
     2401 tttccactct gcccctccca gaggttcagt gttttgtgtc ctctagagtc gacctgcagg
     2461 catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2521 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     2581 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     2641 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2701 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2761 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2821 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2881 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2941 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3001 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3061 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3121 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3181 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3241 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3301 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3361 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3421 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3481 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3541 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3601 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3661 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3721 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3781 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3841 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3901 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3961 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4021 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4081 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4141 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4201 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4261 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4321 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4381 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4441 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4501 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4561 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4621 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     4681 aggcgtatca cgaggccctt tcgtc
//
