LOCUS       sequence-231651-        7443 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 7443)
  TITLE     Gadella lab plasmids
REFERENCE   2  (bases 1 to 7443)
  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 (mScarlet-I_10_LSS-SGFP2_10)
FEATURES             Location/Qualifiers
     source          1..7443
                     /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"
     primer_bind     complement(2559..2577)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2827..2846
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      3272..3281
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3278..3280,3281..3283,3284..3796)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(3323..3344)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3584..3603)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3931..3952
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3971..4549
                     /codon_start=1
                     /product="human Rho family small GTPase RhoA"
                     /label=RhoA
                     /translation="AAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADI
                     EVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKH
                     FCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTK
                     DGVREVFEMATRAALQARRGKKKSGCLVL"
     CDS             4586..4618
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             4625..4648
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4738..4757)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4803..4858
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4857..4876)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4946..4993
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5006..5711
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5094..5466
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5123..5711)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5720..5753)
                     /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(5921..6508)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6000..6019)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6521..7312,7313..7381))
                     /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     7144..7163
                     /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 ggaccggtcg ccaccatggt gagcaagggc gaggcagtga tcaaggagtt
     2461 catgcggttc aaggtgcaca tggagggctc catgaacggc cacgagttcg agatcgaggg
     2521 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     2581 tggccccctg cccttctcct gggacatcct gtcccctcag ttcatgtacg gctccagggc
     2641 cttcatcaag caccccgccg acatccccga ctactataag cagtccttcc ccgagggctt
     2701 caagtgggag cgcgtgatga acttcgagga cggcggcgcc gtgaccgtga cccaggacac
     2761 ctccctggag gacggcaccc tgatctacaa ggtgaagctc cgcggcacca acttccctcc
     2821 tgacggcccc gtaatgcaga agaagacaat gggctgggaa gcgtccaccg agcggttgta
     2881 ccccgaggac ggcgtgctga agggcgacat taagatggcc ctgcgcctga aggacggcgg
     2941 ccgctacctg gcggacttca agaccaccta caaggccaag aagcccgtgc agatgcccgg
     3001 cgcctacaac gtcgaccgca agttggacat cacctcccac aacgaggact acaccgtggt
     3061 ggaacagtac gaacgctccg agggccgcca ctgtacggag gcacaaaagc agaaagagca
     3121 gagacaggcc gcagaggagc tggccaacgg ctctgaagcc cagaagcaaa aggaacagcg
     3181 ccaggcagca gaggaactgg caaacggaag cgaagcacag aaacagaagg aacagagaca
     3241 ggcagccgaa gagctggcaa aaacgccggt cgccaccatg gtgagcaagg gcgaggagct
     3301 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     3361 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     3421 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgagctacgg
     3481 cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     3541 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     3601 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     3661 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     3721 ccacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat
     3781 ccgccacaac atcgaggacg gcggcgtgca gctcgccgac cactaccagc agaacacccc
     3841 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcatcc agtccaagct
     3901 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     3961 cgggtgtacc gctgccatcc ggaagaaact ggtgattgtt ggtgatggag cctgtggaaa
     4021 gacatgcttg ctcatagtct tcagcaagga ccagttccca gaggtgtatg tgcccacagt
     4081 gtttgagaac tatgtggcag atatcgaggt ggatggaaag caggtagagt tggctttgtg
     4141 ggacacagct gggcaggaag attatgatcg cctgaggccc ctctcctacc cagataccga
     4201 tgttatactg atgtgttttt ccatcgacag ccctgatagt ttagaaaaca tcccagaaaa
     4261 gtggacccca gaagtcaagc atttctgtcc caacgtgccc atcatcctgg ttgggaataa
     4321 gaaggatctt cggaatgatg agcacacaag gcgggagctt gccaagatga agcaggagcc
     4381 ggtgaaacct gaagaaggca gagatatggc aaacaggatt ggcgcttttg ggtacatgga
     4441 gtgttcagca aagaccaaag atggagtgag agaggttttt gaaatggcta cgagagctgc
     4501 tctgcaagct agacgtggga agaaaaaatc tgggtgcctt gtcttgtgaa agcttgcggc
     4561 cgcacagctg tatacacgtg caagccagcc agaactcgcc ccggaagacc ccgaggatct
     4621 cgagcaccac catcaccatc accatcacta agtgattaac ctcaggtgca ggctgcctat
     4681 cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcgatct
     4741 ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg
     4801 ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact
     4861 cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag
     4921 tttggcaaca tatgcccata tgtaactagc ataacccctt ggggcctcta aacgggtctt
     4981 gaggggtttt ttgctgaaag catgcggagg aaattctcct tgaagtttcc ctggtgttca
     5041 aagtaaagga gtttgcacca gacgcacctc tgttcactgg tccggcgtat taaaacacga
     5101 tacattgtta ttagtacatt tattaagcgc tagattctgt gcgttgttga tttacagaca
     5161 attgttgtac gtattttaat aattcattaa atttataatc tttagggtgg tatgttagag
     5221 cgaaaatcaa atgattttca gcgtctttat atctgaattt aaatattaaa tcctcaatag
     5281 atttgtaaaa taggtttcga ttagtttcaa acaagggttg tttttccgaa ccgatggctg
     5341 gactatctaa tggattttcg ctcaacgcca caaaacttgc caaatcttgt agcagcaatc
     5401 tagctttgtc gatattcgtt tgtgttttgt tttgtaataa aggttcgacg tcgttcaaaa
     5461 tattatgcgc ttttgtattt ctttcatcac tgtcgttagt gtacaattga ctcgacgtaa
     5521 acacgttaaa tagagcttgg acatatttaa catcgggcgt gttagcttta ttaggccgat
     5581 tatcgtcgtc gtcccaaccc tcgtcgttag aagttgcttc cgaagacgat tttgccatag
     5641 ccacacgacg cctattaatt gtgtcggcta acacgtccgc gatcaaattt gtagttgagc
     5701 tttttggaat tgcgatcgca taacttcgta tagcatacat tatacgaagt tataagctcg
     5761 gaacgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5821 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5881 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5941 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6001 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6061 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6121 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6181 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6241 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6301 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6361 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6421 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6481 gattacgcgc agaaaaaaag gatctcaaga agatcctttg ttaccaatgc ttaatcagtg
     6541 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6601 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6661 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6721 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6781 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6841 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6901 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6961 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7021 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7081 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7141 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7201 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7261 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7321 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7381 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgtccgcgc
     7441 gtt
//
