LOCUS       sequence-247562-        7500 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 7500)
  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 7500)
  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: pTriExRhoA2G
FEATURES             Location/Qualifiers
     source          1..7500
                     /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             join(3371..3373,3374..3376,3377..4087)
                     /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(3416..3437)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3677..3696)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4024..4045
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4682..4705
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4795..4814)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4860..4915
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4914..4933)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5003..5050
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5063..5768
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5151..5523
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5180..5768)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5777..5810)
                     /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(5978..6565)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6057..6076)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6578..7369,7370..7438))
                     /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     7201..7220
                     /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 atccggcggc caccaccgcg ttgacttcaa
     2221 gaccatctac agggccaaga aggcggtgaa gctgcccgac tatcactttg tggaccaccg
     2281 catcgagatc ctgaaccacg acaaggacta caacaaggtg accgtttacg agagcgccgt
     2341 ggcccgcaac tccaccgacg gcatggacga gctgtacaag ggtgctagcg gaggaatggt
     2401 gagcaagggc gaggagacca caatgggcgt aatcaagccc gacatgaaga tcaagctgaa
     2461 gatggagggc aacgtgaatg gccacgcctt cgtgatcgag ggcgagggcg agggcaagcc
     2521 ctacgacggc accaacacca tcaacctgga ggtgaaggag ggagcccccc tgcccttctc
     2581 ctacgacatt ctgaccaccg cgttcgccta cggcaacagg gccttcacca agtaccccga
     2641 cgacatcccc aactacttca agcagtcctt ccccgagggc tactcttggg agcgcaccat
     2701 gaccttcgag gacaagggca tcgtgaaggt gaagtccgac atctccatgg aggaggactc
     2761 cttcatctac gagatacacc tcaagggcga gaacttcccc cccaacggcc ccgtgatgca
     2821 gaagaagacc accggctggg acgcctccac cgagaggatg tacgtgcgcg acggcgtgct
     2881 gaagggcgac gtcaagcaca agctgctgct ggagggctcc ggaatcctgg aggacctcaa
     2941 tatgctctac atccggcaga tggcactcag cctggaggac acagagctgc agaggaaact
     3001 agatcatgag atccgcatga gggatggggc ctgcaagctg ctggcagcct gctcccagcg
     3061 agagcaggct ctggaagcca ccaagagcct gctggtgtgc aacagccgta ttctcagcta
     3121 catgggtgag ctgcagcggc gaaaggaggc ccaggtgctg gagaagacac ccgggacttc
     3181 tggttctggt aaacctggtt ctggtgaagg ttctactaaa acttctggtt ctggtaaacc
     3241 tggttctggt gaaggttcta ctaaaacttc tggttctggt aaacctggtt ctggtgaagg
     3301 ttctactaaa acttctggtt ctggtaaacc tggttctggt gaaggttcta ctaaaaccgg
     3361 tgcggccgcg atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     3421 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     3481 tgccacctac ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc
     3541 ctggcccacc ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga
     3601 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     3661 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     3721 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3781 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa
     3841 gcagaagaac ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt
     3901 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3961 cgacaaccac tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga
     4021 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     4081 gtacaagggt accgctgcca ttcgtaagaa actggtgatt gttggtgatg gagcctgtgg
     4141 aaagacatgc ttgctcatag tcttcagcaa ggaccagttc ccagaggtgt atgtgcccac
     4201 agtgtttgag aactatgtgg cagatatcga ggtggatgga aagcaggtag agttggcttt
     4261 gtgggacaca gctgggcagg aagattatga tcgcctgagg cccctctcct acccagatac
     4321 cgatgttata ctgatgtgtt tttccatcga cagccctgat agtttagaaa acatcccaga
     4381 aaagtggacc ccagaagtca agcatttctg tcccaacgtg cccatcatcc tggttgggaa
     4441 taagaaggat cttcggaatg atgagcacac aaggcgggag ctagccaaga tgaagcagga
     4501 gccggtgaaa cctgaagaag gcagagatat ggcaaacagg attggcgctt ttgggtacat
     4561 ggagtgttca gcaaagacca aagatggagt gagagaggtt tttgaaatgg ctacgagagc
     4621 tgctctgcaa gctagacgtg ggaagaaaaa atctggttgc cttgtcttgt gataactcga
     4681 gcaccaccat caccatcacc atcactaagt gattaacctc aggtgcaggc tgcctatcag
     4741 aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga tcgatctttt
     4801 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta
     4861 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg
     4921 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt
     4981 ggcaacatat gcccatatgt aactagcata accccttggg gcctctaaac gggtcttgag
     5041 gggttttttg ctgaaagcat gcggaggaaa ttctccttga agtttccctg gtgttcaaag
     5101 taaaggagtt tgcaccagac gcacctctgt tcactggtcc ggcgtattaa aacacgatac
     5161 attgttatta gtacatttat taagcgctag attctgtgcg ttgttgattt acagacaatt
     5221 gttgtacgta ttttaataat tcattaaatt tataatcttt agggtggtat gttagagcga
     5281 aaatcaaatg attttcagcg tctttatatc tgaatttaaa tattaaatcc tcaatagatt
     5341 tgtaaaatag gtttcgatta gtttcaaaca agggttgttt ttccgaaccg atggctggac
     5401 tatctaatgg attttcgctc aacgccacaa aacttgccaa atcttgtagc agcaatctag
     5461 ctttgtcgat attcgtttgt gttttgtttt gtaataaagg ttcgacgtcg ttcaaaatat
     5521 tatgcgcttt tgtatttctt tcatcactgt cgttagtgta caattgactc gacgtaaaca
     5581 cgttaaatag agcttggaca tatttaacat cgggcgtgtt agctttatta ggccgattat
     5641 cgtcgtcgtc ccaaccctcg tcgttagaag ttgcttccga agacgatttt gccatagcca
     5701 cacgacgcct attaattgtg tcggctaaca cgtccgcgat caaatttgta gttgagcttt
     5761 ttggaattgc gatcgcataa cttcgtatag catacattat acgaagttat aagctcggaa
     5821 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5881 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5941 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgccccctga
     6001 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     6061 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6121 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6181 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6241 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6301 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6361 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6421 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6481 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6541 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgtta ccaatgctta atcagtgagg
     6601 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6661 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6721 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6781 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6841 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6901 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6961 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7021 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7081 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7141 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7201 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7261 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7321 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7381 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7441 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg tccgcgcgtt
//
