LOCUS       sequence-50305-e        7034 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 7034)
  AUTHORS   Fritz RD, Letzelter M, Reimann A, Martin K, Fusco L, Ritsma L, 
            Ponsioen B, Fluri E, Schulte-Merker S, van Rheenen J, Pertz O
  TITLE     A Versatile Toolkit to Produce Sensitive FRET Biosensors to 
            Visualize Signaling in Time and Space.
  JOURNAL   Sci Signal. 2013 Jul 23;6(285):rs12. doi: 10.1126/scisignal.2004135.
  PUBMED    23882122
REFERENCE   2  (bases 1 to 7034)
  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: "Sci 
            Signal."; date: "2013-07-23"; volume: "6(285):rs12. doi"; pages: " 
            10.1126/scisignal.2004135"
COMMENT     Sequence Label: EKAR2G_design1_mTFP_wt_Venus_wt
FEATURES             Location/Qualifiers
     source          1..7034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1076..1379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1381..1583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1534..1554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1786..1808
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1870..1889
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1931..1950
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1931..1949
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1950..1974
                     /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        1990..2099
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             2120..2137
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2147..2176
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2195..2899
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="VSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDGT
                     NTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTFE
                     DKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLKG
                     DVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESAV
                     ARNSTDGMDELYK"
     CDS             join(3479..3481,3482..3484,3485..4195)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3524..3545)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3785..3804)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4132..4153
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4265..4288
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     polyA_signal    4394..4449
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4448..4467)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4537..4584
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4597..5302
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4685..5057
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4714..5302)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5311..5344)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(5512..6099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5591..5610)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6112..6903,6904..6972))
                     /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     6735..6754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggcct
     1021 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1081 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1141 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1201 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc
     1261 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1321 ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc
     1381 tgatgcggtt ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc
     1441 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1501 ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt
     1561 gggaggtcta tataagcaga cgtcgtttag tgaaccgtca gatcactaga tgctttattg
     1621 cggtagttta tcacagttaa attgctaacg ccagtctcga acttaacgtg cagaagttgg
     1681 tcgtgaggca ctgggcaggt aagtatcggg ccctttgtgc ggggggagcg gctcggggct
     1741 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1801 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1861 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc
     1921 ggaccgaaat taatacgact cactataggg gaattgtgag cggataacaa ttccccggag
     1981 ttaatccggg acctttaatt caacccaaca caatatatta tagttaaata agaattatta
     2041 tcaaatcatt tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca
     2101 aaggagatat accatggcac accatcacca ccaccacggg agcggggagc agaaactaat
     2161 atcagaggag gatcttggct caggttcagg atccgtgagc aagggcgagg agaccacaat
     2221 gggcgtaatc aagcccgaca tgaagatcaa gctgaagatg gagggcaacg tgaatggcca
     2281 cgccttcgtg atcgagggcg agggcgaggg caagccctac gacggcacca acaccatcaa
     2341 cctggaggtg aaggagggag cccccctgcc cttctcctac gacattctga ccaccgcgtt
     2401 cgcctacggc aacagggcct tcaccaagta ccccgacgac atccccaact acttcaagca
     2461 gtccttcccc gagggctact cttgggagcg caccatgacc ttcgaggaca agggcatcgt
     2521 gaaggtgaag tccgacatct ccatggagga ggactccttc atctacgaga tacacctcaa
     2581 gggcgagaac ttccccccca acggccccgt gatgcagaag aagaccaccg gctgggacgc
     2641 ctccaccgag aggatgtacg tgcgcgacgg cgtgctgaag ggcgacgtca agcacaagct
     2701 gctgctggag ggcggcggcc accaccgcgt tgacttcaag accatctaca gggccaagaa
     2761 ggcggtgaag ctgcccgact atcactttgt ggaccaccgc atcgagatcc tgaaccacga
     2821 caaggactac aacaaggtga ccgtttacga gagcgccgtg gcccgcaact ccaccgacgg
     2881 catggacgag ctgtacaagt ccggaatggc ggacgaggag aagctgccgc ccggctggga
     2941 gaagcgcatg agccgcagct caggccgagt gtactacttc aaccacatca ctaacgccag
     3001 ccagtgggag cggcccagcg gcaacagcag cagtggtggc aaaaacgggc agggggagcc
     3061 tgccaggaag ctgcgcattc tgcagtcgac tagggagcaa gggcttgtgc ccgggtctag
     3121 aggcggaggt ggtggaggag gtggaggtgg tggaggtgga ggtggaggtg gtggcggtgg
     3181 tggaggagga ggagctagag gcggaggtgg tggaggaggt ggaggtggtg gaggtggagg
     3241 tggaggtggt ggcggtggtg gaggaggagg agccagcggc ggaggtggtg gaggaggtgg
     3301 aggtggtgga ggtggaggtg gaggtggtgg cggtggtgga ggaggaggaa ccggtgctga
     3361 ccaactgact gaagagtggt cgacggtaca gcggaccgcg cctgacgtac cacgtacacc
     3421 agtaggaaag gcaaagcttt cattccaatt cccgcgggcg cgggacatcg cggccgcgat
     3481 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     3541 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     3601 caagctgacc ctgaagctga tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     3661 cgtgaccacc ctgggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca
     3721 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     3781 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     3841 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     3901 gctggagtac aactacaaca gccacaacgt ctatatcacc gccgacaagc agaagaacgg
     3961 catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga
     4021 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     4081 cctgagctac cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     4141 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggtac
     4201 cgatatcctt caaaaaaaac ttgaagaact tgaacttgac gaatgagata tctaatgact
     4261 cgagcaccac catcaccatc accatcacta agtgattaac ctcaggtgca ggctgcctat
     4321 cagaaggtgg tggctggtgt ggccaaaaat tatggggaca tcatgaagcc ccttgagcat
     4381 ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg
     4441 tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat
     4501 ttggtttaga gtttggcaac atatgcccat atgtaactag cataacccct tggggcctct
     4561 aaacgggtct tgaggggttt tttgctgaaa gcatgcggag gaaattctcc ttgaagtttc
     4621 cctggtgttc aaagtaaagg agtttgcacc agacgcacct ctgttcactg gtccggcgta
     4681 ttaaaacacg atacattgtt attagtacat ttattaagcg ctagattctg tgcgttgttg
     4741 atttacagac aattgttgta cgtattttaa taattcatta aatttataat ctttagggtg
     4801 gtatgttaga gcgaaaatca aatgattttc agcgtcttta tatctgaatt taaatattaa
     4861 atcctcaata gatttgtaaa ataggtttcg attagtttca aacaagggtt gtttttccga
     4921 accgatggct ggactatcta atggattttc gctcaacgcc acaaaacttg ccaaatcttg
     4981 tagcagcaat ctagctttgt cgatattcgt ttgtgttttg ttttgtaata aaggttcgac
     5041 gtcgttcaaa atattatgcg cttttgtatt tctttcatca ctgtcgttag tgtacaattg
     5101 actcgacgta aacacgttaa atagagcttg gacatattta acatcgggcg tgttagcttt
     5161 attaggccga ttatcgtcgt cgtcccaacc ctcgtcgtta gaagttgctt ccgaagacga
     5221 ttttgccata gccacacgac gcctattaat tgtgtcggct aacacgtccg cgatcaaatt
     5281 tgtagttgag ctttttggaa ttgcgatcgc ataacttcgt atagcataca ttatacgaag
     5341 ttataagctc ggaacgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5401 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5461 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5521 gctccgcccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5581 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5641 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5701 tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5761 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5821 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5881 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5941 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6001 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6061 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gttaccaatg
     6121 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6181 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6241 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6301 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6361 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6421 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6481 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6541 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6601 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6661 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6721 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6781 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6841 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6901 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6961 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7021 catgtccgcg cgtt
//
