LOCUS       sequence-220190-        7491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RhoA FRET biosensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7491)
  TITLE     Gadella lab plasmids
REFERENCE   2  (bases 1 to 7491)
  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"
COMMENT     Sequence Label: RhoA sensor (LSS-SGFP2-mCherry)
FEATURES             Location/Qualifiers
     source          1..7491
                     /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"
     regulatory      2420..2429
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2426..3133
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2571..2589)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2839..2858
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(3329..3331,3332..3844)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIE"
     primer_bind     complement(3371..3392)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3632..3651)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3979..4000
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4019..4597
                     /codon_start=1
                     /product="human Rho family small GTPase RhoA"
                     /label=RhoA
                     /translation="AAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADI
                     EVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKH
                     FCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTK
                     DGVREVFEMATRAALQARRGKKKSGCLVL"
     CDS             4634..4666
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             4673..4696
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4786..4805)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4851..4906
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4905..4924)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4994..5041
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5054..5759
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5142..5514
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5171..5759)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5768..5801)
                     /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(5969..6556)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6048..6067)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6569..7360,7361..7429))
                     /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     7192..7211
                     /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 ccatcacgga tctggttcta gcctgggccc
     2161 cgtagagctg ctgctgcggg gctcctcgcg ccgcctcgac ctgctgcacc agcagctgca
     2221 ggagctgcac gcccacggtt ctggttccct gctagagcag ctgggcctgg ccggggcaga
     2281 cctggcggcc cccggggtac agcagcagct ggagctggag cgggagcggc tgcggcggga
     2341 aatccgcaag gagctgaagc tgaaggaggg tgctgagaac ctgcggcggg ccaccactga
     2401 cctgggccgc ggtaccggtg ccaccatggt gagcaagggc gaggaggata acatggccat
     2461 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     2521 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     2581 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     2641 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     2701 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     2761 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     2821 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc
     2881 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     2941 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     3001 gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga
     3061 ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga
     3121 cgagctgtac aagtccggac tcagatctaa catcgaggaa gcacaaaagc agaaggaaca
     3181 gagacaggca gcagaggagc tggcaaacgg atctgaagca caaaagcaga aggaacagag
     3241 acaggcagca gaggagctgg caaacggatc tgaagcacaa aagcagaagg aacagagaca
     3301 ggcagcagag gagctggcaa acggatccgt gagcaagggc gaggagctgt tcaccggggt
     3361 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     3421 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     3481 caagctgccc gtgccctggc ccaccctcgt gaccaccctg agctacggcg tgcagtgctt
     3541 cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     3601 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     3661 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3721 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     3781 tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat
     3841 cgaggacggc ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3901 ccccgtgctg ctgcccgaca accactacct gagcatccag tccaagctga gcaaagaccc
     3961 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg gggctagcgc
     4021 tgccatccgg aagaaactgg tgattgttgg tgatggagcc tgtggaaaga catgcttgct
     4081 catagtcttc agcaaggacc agttcccaga ggtgtatgtg cccacagtgt ttgagaacta
     4141 tgtggcagat atcgaggtgg atggaaagca ggtagagttg gctttgtggg acacagctgg
     4201 gcaggaagat tatgatcgcc tgaggcccct ctcctaccca gataccgatg ttatactgat
     4261 gtgtttttcc atcgacagcc ctgatagttt agaaaacatc ccagaaaagt ggaccccaga
     4321 agtcaagcat ttctgtccca acgtgcccat catcctggtt gggaataaga aggatcttcg
     4381 gaatgatgag cacacaaggc gggagcttgc caagatgaag caggagccgg tgaaacctga
     4441 agaaggcaga gatatggcaa acaggattgg cgcttttggg tacatggagt gttcagcaaa
     4501 gaccaaagat ggagtgagag aggtttttga aatggctacg agagctgctc tgcaagctag
     4561 acgtgggaag aaaaaatctg ggtgccttgt cttgtgaaag cttgcggccg cacagctgta
     4621 tacacgtgca agccagccag aactcgcccc ggaagacccc gaggatctcg agcaccacca
     4681 tcaccatcac catcactaag tgattaacct caggtgcagg ctgcctatca gaaggtggtg
     4741 gctggtgtgg ccaatgccct ggctcacaaa taccactgag atcgatcttt ttccctctgc
     4801 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     4861 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata
     4921 tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata
     4981 tgcccatatg taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     5041 gctgaaagca tgcggaggaa attctccttg aagtttccct ggtgttcaaa gtaaaggagt
     5101 ttgcaccaga cgcacctctg ttcactggtc cggcgtatta aaacacgata cattgttatt
     5161 agtacattta ttaagcgcta gattctgtgc gttgttgatt tacagacaat tgttgtacgt
     5221 attttaataa ttcattaaat ttataatctt tagggtggta tgttagagcg aaaatcaaat
     5281 gattttcagc gtctttatat ctgaatttaa atattaaatc ctcaatagat ttgtaaaata
     5341 ggtttcgatt agtttcaaac aagggttgtt tttccgaacc gatggctgga ctatctaatg
     5401 gattttcgct caacgccaca aaacttgcca aatcttgtag cagcaatcta gctttgtcga
     5461 tattcgtttg tgttttgttt tgtaataaag gttcgacgtc gttcaaaata ttatgcgctt
     5521 ttgtatttct ttcatcactg tcgttagtgt acaattgact cgacgtaaac acgttaaata
     5581 gagcttggac atatttaaca tcgggcgtgt tagctttatt aggccgatta tcgtcgtcgt
     5641 cccaaccctc gtcgttagaa gttgcttccg aagacgattt tgccatagcc acacgacgcc
     5701 tattaattgt gtcggctaac acgtccgcga tcaaatttgt agttgagctt tttggaattg
     5761 cgatcgcata acttcgtata gcatacatta tacgaagtta taagctcgga acgctgcgct
     5821 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5881 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5941 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgccccctg acgagcatca
     6001 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6061 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6121 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6181 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6241 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6301 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6361 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6421 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6481 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6541 aaaaaaagga tctcaagaag atcctttgtt accaatgctt aatcagtgag gcacctatct
     6601 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6661 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6721 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6781 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6841 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6901 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6961 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7021 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7081 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7141 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7201 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7261 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7321 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7381 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7441 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gtccgcgcgt t
//
