LOCUS       sequence-50767-e        6888 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 6888)
  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 6888)
  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_design2_mTFP_wt_Venus_wt
FEATURES             Location/Qualifiers
     source          1..6888
                     /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             2414..3118
                     /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(3179..3181,3182..3184,3185..3895)
                     /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(3224..3245)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3485..3504)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3832..3853
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4070..4093
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4183..4202)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4248..4303
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4302..4321)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4391..4438
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4451..5156
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4539..4911
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4568..5156)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5165..5198)
                     /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(5366..5953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5445..5464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5966..6757,6758..6826))
                     /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     6589..6608
                     /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 ccatcacggg agcggggagc agaaactaat
     2161 atcagaggag gatcttggct caggaaccgg tagatctggg cacatggcgg acgaggagaa
     2221 gctgccgccc ggctgggaga agcgcatgag ccgcagctca ggccgagtgt actacttcaa
     2281 ccacatcact aacgccagcc agtgggagcg gcccagcggc aacagcagca gtggtggcaa
     2341 aaacgggcag ggggagcctg ccaggaagct gcgcattctg cagtcgacta gggagcaagg
     2401 gcttgtggga tccgtgagca agggcgagga gaccacaatg ggcgtaatca agcccgacat
     2461 gaagatcaag ctgaagatgg agggcaacgt gaatggccac gccttcgtga tcgagggcga
     2521 gggcgagggc aagccctacg acggcaccaa caccatcaac ctggaggtga aggagggagc
     2581 ccccctgccc ttctcctacg acattctgac caccgcgttc gcctacggca acagggcctt
     2641 caccaagtac cccgacgaca tccccaacta cttcaagcag tccttccccg agggctactc
     2701 ttgggagcgc accatgacct tcgaggacaa gggcatcgtg aaggtgaagt ccgacatctc
     2761 catggaggag gactccttca tctacgagat acacctcaag ggcgagaact tcccccccaa
     2821 cggccccgtg atgcagaaga agaccaccgg ctgggacgcc tccaccgaga ggatgtacgt
     2881 gcgcgacggc gtgctgaagg gcgacgtcaa gcacaagctg ctgctggagg gcggcggcca
     2941 ccaccgcgtt gacttcaaga ccatctacag ggccaagaag gcggtgaagc tgcccgacta
     3001 tcactttgtg gaccaccgca tcgagatcct gaaccacgac aaggactaca acaaggtgac
     3061 cgtttacgag agcgccgtgg cccgcaactc caccgacggc atggacgagc tgtacaagtc
     3121 cggaacttct ggttctggta aacctggttc tggtgaaggt tctactaaag cggccgcgat
     3181 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     3241 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     3301 caagctgacc ctgaagctga tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     3361 cgtgaccacc ctgggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca
     3421 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     3481 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     3541 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     3601 gctggagtac aactacaaca gccacaacgt ctatatcacc gccgacaagc agaagaacgg
     3661 catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga
     3721 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     3781 cctgagctac cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     3841 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggtac
     3901 cgctgaccaa ctgactgaag agtggtcgac ggtacagcgg accgcgcctg acgtaccacg
     3961 tacaccagta ggaaaggcaa agctttcatt ccaattcccg cgggcgcggg atatccttca
     4021 aaaaaaactt gaagaacttg aacttgacga atgagatatc tgactcgagc accaccatca
     4081 ccatcaccat cactaagtga ttaacctcag gtgcaggctg cctatcagaa ggtggtggct
     4141 ggtgtggcca atgccctggc tcacaaatac cactgagatc gatctttttc cctctgccaa
     4201 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     4261 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     4321 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     4381 ccatatgtaa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     4441 gaaagcatgc ggaggaaatt ctccttgaag tttccctggt gttcaaagta aaggagtttg
     4501 caccagacgc acctctgttc actggtccgg cgtattaaaa cacgatacat tgttattagt
     4561 acatttatta agcgctagat tctgtgcgtt gttgatttac agacaattgt tgtacgtatt
     4621 ttaataattc attaaattta taatctttag ggtggtatgt tagagcgaaa atcaaatgat
     4681 tttcagcgtc tttatatctg aatttaaata ttaaatcctc aatagatttg taaaataggt
     4741 ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat ggctggacta tctaatggat
     4801 tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag caatctagct ttgtcgatat
     4861 tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg
     4921 tatttctttc atcactgtcg ttagtgtaca attgactcga cgtaaacacg ttaaatagag
     4981 cttggacata tttaacatcg ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc
     5041 aaccctcgtc gttagaagtt gcttccgaag acgattttgc catagccaca cgacgcctat
     5101 taattgtgtc ggctaacacg tccgcgatca aatttgtagt tgagcttttt ggaattgcga
     5161 tcgcataact tcgtatagca tacattatac gaagttataa gctcggaacg ctgcgctcgg
     5221 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5281 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5341 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg ccccctgacg agcatcacaa
     5401 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5461 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5521 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct
     5581 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5641 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5701 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5761 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5821 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5881 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5941 aaaaggatct caagaagatc ctttgttacc aatgcttaat cagtgaggca cctatctcag
     6001 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6061 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6121 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6181 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6241 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6301 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6361 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6421 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6481 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6541 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6601 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6661 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6721 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6781 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6841 aatattattg aagcatttat cagggttatt gtctcatgtc cgcgcgtt
//
