LOCUS       sequence-369482-        4861 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template for CD3-epsilon-mNeonGreen knock-in at C-terminus of 
            human EGFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4861)
  AUTHORS   Farahani PE, Yang X, Mesev EV, Fomby KA, Brumbaugh-Reed EH, Bashor 
            CJ, Nelson CM, Toettcher JE
  TITLE     pYtags enable spatiotemporal measurements of receptor tyrosine 
            kinase signaling in living cells.
  JOURNAL   eLife. 2023 May 22;12:e82863. doi: 10.7554/eLife.82863.
  PUBMED    37212240
REFERENCE   2  (bases 1 to 4861)
  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: "eLife."; 
            date: "2023-05-22"; volume: "12:e82863. doi"; pages: " 
            10.7554/eLife.82863"
COMMENT     Sequence Label: pUC19 EGFRup-CD3-epsilon-mNeonGreen-EGFRdown
FEATURES             Location/Qualifiers
     source          1..4861
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             734..1441
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      986..995
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2336..2358
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2396..2414)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2482..2586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2587..2655,2656..3447)
                     /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"
     rep_origin      3618..4206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4107..4126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4360..4377
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccagggatcc tgcatgggat ggtgctttgc tgattacttc acctctgatt tctttccact
       61 ttcagaatac ataaaccagt ccgttcccaa aaggcccgct ggctctgtgc agaatcctgt
      121 ctatcacaat cagcctctga accccgcgcc cagcagagac ccacactacc aggaccccca
      181 cagcactgca gtgggcaacc ccgagtatct caacactgtc cagcccacct gtgtcaacag
      241 cacattcgac agccctgccc actgggccca gaaaggcagc caccaaatta gcctggacaa
      301 ccctgactac cagcaggact tctttcccaa ggaagccaag ccaaatggca tctttaaggg
      361 ctccacagct gaaaatgcag aatacctaag ggtcgcgcca caaagcagtg aatttattgg
      421 agcaggctct ggttctccac ctcccgtccc taatcccgat tatgagccca ttcggaaagg
      481 tcaaagagat ttgtatagtg gcctcaatca aagaagaatc gggtcaggtt ccggcccgcc
      541 gccagtccca aaccccgact atgaaccaat caggaaaggc caacgggatt tgtactcagg
      601 attgaatcag cgcaggatag gttccggctc cgggccacca cccgtcccta acccagatta
      661 cgaacccata cgcaagggac aacgcgatct ttacagcgga ttgaaccaac gacgaatagg
      721 atctggttct ggcgtgagca agggcgagga ggataacatg gcctctctcc cagcgacaca
      781 tgagttacac atctttggct ccatcaacgg tgtggacttt gacatggtgg gtcagggcac
      841 cggcaatcca aatgatggtt atgaggagtt aaacctgaag tccaccaagg gtgacctcca
      901 gttctccccc tggattctgg tccctcatat cgggtatggc ttccatcagt acctgcccta
      961 ccctgacggg atgtcgcctt tccaggccgc catggtagat ggctccggat accaagtcca
     1021 tcgcacaatg cagtttgaag atggtgcctc ccttactgtt aactaccgct acacctacga
     1081 gggaagccac atcaaaggag aggcccaggt gaaggggact ggtttccctg ctgacggtcc
     1141 tgtgatgacc aactcgctga ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa
     1201 cgacaaaacc atcatcagta cctttaagtg gagttacacc actggaaatg gcaagcgcta
     1261 ccggagcact gcgcggacca cctacacctt tgccaagcca atggcggcta actatctgaa
     1321 gaaccagccg atgtacgtgt tccgtaagac ggagctcaag cactccaaga ccgagctcaa
     1381 cttcaaggag tggcaaaagg cctttaccga tgtgatgggc atggacgagc tgtacaagta
     1441 accacggagg atagtatgag ccctaaaaat ccagactctt tcgataccca gcaccaagcc
     1501 acagcaggtc ctccatccca acagccatgc ccgcattagc tcttagaccc acagactggt
     1561 tttgcaacgt ttacaccgac tagccaggaa gtacttccac ctcgggcaca ttttgggaag
     1621 ttgcattcct ttgtcttcaa actgtgaagc atttacagaa acgcatccag caagaatatt
     1681 gtccctttga gcagaaattt atctttcaaa gaggtatatt tgaaaaaata aaaaagtata
     1741 tgtgaggatt tttattgatt ggggatcttg gagtttttca ttgtcgctat tgatttttac
     1801 ttcaatgggc tcttccaaca aggaagaagc ttgctggtag cacttgctac cctgagttca
     1861 tccaggccca actgtgagca aggagcacaa gccacaagtc ttccagagga tgcttgattc
     1921 cagtggttct gcttcaaggc ttccactgca aaacactaaa gatccaagaa ggccttcatg
     1981 gccccagcag gccggatcgg tactgtatca agtcatggca ggtacagtag gataagccac
     2041 tctgtccctt cctgggcaaa gaagaaacgg aggggatgga attcttcctt agacttactt
     2101 ttgtaaaaat gtccccacgg tacttactcc ccactgatgg accagtggtt tccagtcatg
     2161 agcgttagac tgacttgttt gtcttccatt ccattgtttt gaaactcagt atgctgcccc
     2221 tgtcttgctg tcatgaaatc attaagccag ccccgacacc cgccaacacc cgctgacgcg
     2281 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2341 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     2401 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     2461 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2521 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2581 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2641 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2701 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2761 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2821 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2881 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2941 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3001 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3061 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3121 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3181 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3241 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3301 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3361 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3421 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3481 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3541 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3601 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3661 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3721 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     3781 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3841 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3901 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3961 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4021 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4081 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4141 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4201 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4261 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4321 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4381 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4441 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcatata cacacacgta
     4501 cacacacacc acacacaccc acatgcacac accacacaca catacaaata tacaccacac
     4561 acacatacac cacacacacg gtgcacatac acacacatat acacacacca gacacacata
     4621 ccacatacac atcacacata tatgtataca tgcatacaca tacacacaca catacacaca
     4681 ctctcctcaa ggcagtttat cctctgagaa ctttaaattt acaaaagaca catatgtcca
     4741 ttactttgag aaggacagga aagaacccac tttcttttgc agcaacagca agagggccct
     4801 cccgaggctc ctgctccctg tcataagtct ccttgttgag gacattcaca gggttcagaa
     4861 c
//
