LOCUS       sequence-51062-e        7119 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 7119)
  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 7119)
  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: pTriExEKAR2G2
FEATURES             Location/Qualifiers
     source          1..7119
                     /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"
     primer_bind     3571..3592
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(3650..3652,3653..3655,3656..4168)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(3695..3716)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3956..3975)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4301..4324
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4414..4433)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4479..4534
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4533..4552)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4622..4669
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4682..5387
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4770..5142
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4799..5387)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5396..5429)
                     /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(5597..6184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5676..5695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6197..6988,6989..7057))
                     /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     6820..6839
                     /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 atccgaggac aagggcatcg tgaaggtgaa
     2221 gtccgacatc tccatggagg aggactcctt catctacgag atacacctca agggcgagaa
     2281 cttccccccc aacggccccg tgatgcagaa gaagaccacc ggctgggacg cctccaccga
     2341 gaggatgtac gtgcgcgacg gcgtgctgaa gggcgacgtc aagcacaagc tgctgctgga
     2401 gggcggcggc caccaccgcg ttgacttcaa gaccatctac agggccaaga aggcggtgaa
     2461 gctgcccgac tatcactttg tggaccaccg catcgagatc ctgaaccacg acaaggacta
     2521 caacaaggtg accgtttacg agagcgccgt ggcccgcaac tccaccgacg gcatggacga
     2581 gctgtacaag ggtgctagcg gaggaatggt gagcaagggc gaggagacca caatgggcgt
     2641 aatcaagccc gacatgaaga tcaagctgaa gatggagggc aacgtgaatg gccacgcctt
     2701 cgtgatcgag ggcgagggcg agggcaagcc ctacgacggc accaacacca tcaacctgga
     2761 ggtgaaggag ggagcccccc tgcccttctc ctacgacatt ctgaccaccg cgttcgccta
     2821 cggcaacagg gccttcacca agtaccccga cgacatcccc aactacttca agcagtcctt
     2881 ccccgagggc tactcttggg agcgcaccat gaccttctcc ggaatggcgg acgaggagaa
     2941 gctgccgccc ggctgggaga agcgcatgag ccgcagctca ggccgagtgt actacttcaa
     3001 ccacatcact aacgccagcc agtgggagcg gcccagcggc aacagcagca gtggtggcaa
     3061 aaacgggcag ggggagcctg ccaggaagct gcgcattctg cagtcgacta gggagcaagg
     3121 gcttgtgccc gggtctagag gcggaggtgg tggaggaggt ggaggtggtg gaggtggagg
     3181 tggaggtggt ggcggtggtg gaggaggagg agctagaggc ggaggtggtg gaggaggtgg
     3241 aggtggtgga ggtggaggtg gaggtggtgg cggtggtgga ggaggaggag ccagcggcgg
     3301 aggtggtgga ggaggtggag gtggtggagg tggaggtgga ggtggtggcg gtggtggagg
     3361 aggaggaacc ggtgctgacc aactgactga agagtggtcg acggtacagc ggaccgcgcc
     3421 tgacgtacca cgtacaccag taggaaaggc aaagctttca ttccaattcc cgcgggcgcg
     3481 ggacatcgcg gccgccatgc tgctgcccga caaccactac ctgagctacc agtccgccct
     3541 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     3601 cgggatcact ctcggcatgg acgagctgta caagggtggc agcggtggca tggtgagcaa
     3661 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3721 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     3781 cctgaagctg atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     3841 cctgggctac ggcctgatgt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     3901 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     3961 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     4021 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     4081 caactacaac agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc
     4141 caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca
     4201 gcagaacacc cccatcggcg acggccccgt gggtaccgat atccttcaaa aaaaacttga
     4261 agaacttgaa cttgacgaat gagatatcta atgactcgag caccaccatc accatcacca
     4321 tcactaagtg attaacctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     4381 aatgccctgg ctcacaaata ccactgagat cgatcttttt ccctctgcca aaaattatgg
     4441 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
     4501 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
     4561 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgta
     4621 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaagcatg
     4681 cggaggaaat tctccttgaa gtttccctgg tgttcaaagt aaaggagttt gcaccagacg
     4741 cacctctgtt cactggtccg gcgtattaaa acacgataca ttgttattag tacatttatt
     4801 aagcgctaga ttctgtgcgt tgttgattta cagacaattg ttgtacgtat tttaataatt
     4861 cattaaattt ataatcttta gggtggtatg ttagagcgaa aatcaaatga ttttcagcgt
     4921 ctttatatct gaatttaaat attaaatcct caatagattt gtaaaatagg tttcgattag
     4981 tttcaaacaa gggttgtttt tccgaaccga tggctggact atctaatgga ttttcgctca
     5041 acgccacaaa acttgccaaa tcttgtagca gcaatctagc tttgtcgata ttcgtttgtg
     5101 ttttgttttg taataaaggt tcgacgtcgt tcaaaatatt atgcgctttt gtatttcttt
     5161 catcactgtc gttagtgtac aattgactcg acgtaaacac gttaaataga gcttggacat
     5221 atttaacatc gggcgtgtta gctttattag gccgattatc gtcgtcgtcc caaccctcgt
     5281 cgttagaagt tgcttccgaa gacgattttg ccatagccac acgacgccta ttaattgtgt
     5341 cggctaacac gtccgcgatc aaatttgtag ttgagctttt tggaattgcg atcgcataac
     5401 ttcgtatagc atacattata cgaagttata agctcggaac gctgcgctcg gtcgttcggc
     5461 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5521 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5581 ccgcgttgct ggcgtttttc cataggctcc gccccctgac gagcatcaca aaaatcgacg
     5641 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5701 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5761 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     5821 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5881 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5941 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6001 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6061 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6121 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6181 tcaagaagat cctttgttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6241 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6301 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6361 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6421 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6481 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6541 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6601 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6661 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6721 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6781 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6841 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6901 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6961 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7021 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7081 gaagcattta tcagggttat tgtctcatgt ccgcgcgtt
//
