LOCUS       sequence-229407-        5043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5043)
  AUTHORS   Karginov AV, Ding F, Kota P, Dokholyan NV, Hahn KM
  TITLE     Engineered allosteric activation of kinases in living cells.
  JOURNAL   Nat Biotechnol. 2010 Jul;28(7):743-7. doi: 10.1038/nbt.1639. Epub 
            2010 Jun 27.
  PUBMED    20581846
REFERENCE   2  (bases 1 to 5043)
  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"; doi: "10.1038/nbt.1639"; 
            journalName: "Nat Biotechnol"; date: "2010-07"; volume: "28"; issue:
            "7"; pages: "743-7"
COMMENT     Sequence Label: pEGFP-FRB
FEATURES             Location/Qualifiers
     source          1..5043
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        419..622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     573..593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      661..670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(667..669,670..672,673..1383)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIDDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(712..733)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(973..992)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1320..1341
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1432..1710
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     polyA_signal    1885..2006
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1922..1941)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1976..1995
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2013..2468)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2150..2171)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2362..2381
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2495..2599
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2601..2958
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2601..2792
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2624..2644)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2809..2944
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2993..3787
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2993..3787
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3047..3066)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3657..3676
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3975..3994)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4019..4066
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4395..4983
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4884..4903
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
       61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      241 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      421 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      481 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      541 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      601 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggtc
      661 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      721 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      781 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      841 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
      901 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      961 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1021 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1081 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1141 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgatgacgg cagcgtgcag
     1201 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1261 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1321 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1381 aagtccggac tcagatctcg agctcaagct tcgaattcaa tggcttctag aatcctctgg
     1441 catgagatgt ggcatgaagg cctggaagag gcatctcgtt tgtactttgg ggaaaggaac
     1501 gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta tgatggaacg gggcccccag
     1561 actctgaagg aaacatcctt taatcaggcc tatggtcgag atttaatgga ggcccaagag
     1621 tggtgcagga agtacatgaa atcagggaat gtcaaggacc tcctccaagc ctgggacctc
     1681 tattatcatg tgttccgacg aatctcaaag actagttaac cgtacgacgt accagactac
     1741 gcataagaaa agtgaggatc caccggatct agataactga tcataatcag ccataccaca
     1801 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     1861 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     1921 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     1981 ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa
     2041 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2101 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     2161 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2221 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     2281 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     2341 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2401 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     2461 gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2521 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2581 atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     2641 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2701 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2761 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     2821 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     2881 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     2941 ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga
     3001 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3061 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3121 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     3181 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3241 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3301 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3361 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3421 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3481 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     3541 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3601 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3661 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3721 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3781 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     3841 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3901 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct
     3961 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     4021 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt
     4081 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
     4141 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
     4201 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt
     4261 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4321 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4381 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4441 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4501 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4561 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4621 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4681 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4741 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4801 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4861 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4921 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4981 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5041 atg
//
