LOCUS       sequence-205449-        6638 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FKBP-EGFP-HOTag3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6638)
  AUTHORS   Zhang Q, Huang H, Lu Q, Wu R, Chung CI, Zhang SQ, Torra J, Schepis 
            A, Coughlin SR, Kornberg TB, Shu X
  TITLE     Visualizing Dynamics of Cell Signaling In Vivo with a Phase 
            Separation-Based Kinase Reporter.
  JOURNAL   Mol Cell. 2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 
            10.1016/j.molcel.2017.12.008.
  PUBMED    29307513
REFERENCE   2  (bases 1 to 6638)
  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: "Mol Cell. 
            2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 
            10.1016/j.molcel.2017.12.008."
COMMENT     Sequence Label: pcDNA3-FKBP-EGFP-HOTag3
FEATURES             Location/Qualifiers
     source          1..6638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        149..528
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        529..732
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     683..703
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     777..796
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        777..795
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      831..840
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(837..839,840..1160)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             join(1203..1205,1206..1208,1209..1919)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1248..1269)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1509..1528)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1856..1877
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2143..2160)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2149..2373
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2419..2847
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2506..2525)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2716..2737
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2856..2876)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2861..3190
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3041..3176
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3103..3122
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3257..4051
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3311..3330)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3921..3940
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4225..4346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4262..4281)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4316..4335
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4395..4411)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4395..4411)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4408..4430)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4419..4435
                     /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(4443..4449,4450..4467,4468..4473))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4488..4509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4626..4643)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4797..5385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4877..4896)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5556..6347,6348..6416))
                     /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     6179..6198
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6417..6521)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6596..6615)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
       61 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
      121 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      181 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      241 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      301 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      361 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      421 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      481 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      541 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      601 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      661 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      721 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
      781 acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt gccaccatgg
      841 gtgtgcaggt agaaacaatc tccccgggag atggccgcac gttccccaag aggggacaga
      901 cctgtgtggt gcactacacc ggtatgctcg aagacggcaa gaagttcgat agctcccgag
      961 accgaaacaa gcccttcaag ttcatgctgg gcaagcaaga ggtcatacgc ggttgggaag
     1021 aaggcgtggc ccagatgagc gtagggcagc gcgccaagct gaccattagc cccgactacg
     1081 cctacggggc caccgggcac cccggcatca ttccacccca tgcgacactc gtctttgatg
     1141 tggagctgct caagctggaa ctcgagtccg gactcagatc tggcagcggt ggaggcgata
     1201 tcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1261 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1321 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1381 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1441 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1501 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1561 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1621 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1681 acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1741 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1801 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1861 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     1921 gatccggcag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggcagtg
     1981 ctggtggtag tgctggtggt agtgctggtg gctctagagg agaaatcgca aagagtctga
     2041 aagaaattgc aaaatccttg aaggaaatag cgtggtcact gaaagaaatt gccaagtccc
     2101 tcaaagggta agaattcggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     2161 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2221 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2281 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2341 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     2401 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2461 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2521 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2581 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2641 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2701 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2761 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2821 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     2881 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2941 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3001 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3061 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3121 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3181 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     3241 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3301 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3361 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3421 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3481 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3541 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3601 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3661 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3721 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3781 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3841 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3901 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3961 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4021 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4081 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4141 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4201 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4261 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4321 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4381 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4441 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4501 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4561 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4621 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4681 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4741 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4801 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4861 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4921 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4981 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5041 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5101 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5161 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5221 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5281 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5341 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5401 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5461 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5521 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5581 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5641 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5701 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5761 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5821 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5881 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5941 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6001 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6061 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6121 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6181 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6241 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6301 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6361 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6421 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6481 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6541 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     6601 tctgctctga tgccgcatag ttaagccagt atctgctc
//
