LOCUS       sequence-156958-        6460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Zdk1 tagged RhoA Q63L.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6460)
  AUTHORS   Wang H, Vilela M, Winkler A, Tarnawski M, Schlichting I, Yumerefendi
            H, Kuhlman B, Liu R, Danuser G, Hahn KM
  TITLE     LOVTRAP: an optogenetic system for photoinduced protein 
            dissociation.
  JOURNAL   Nat Methods. 2016 Jul 18. doi: 10.1038/nmeth.3926.
  PUBMED    27427858
REFERENCE   2  (bases 1 to 6460)
  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: "Nat 
            Methods. 2016 Jul 18. doi: 10.1038/nmeth.3926."
COMMENT     Sequence Label: pTriEx-mVenus-Zdk1-RhoA Q63L
FEATURES             Location/Qualifiers
     source          1..6460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     110..937
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             110..234
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(272..877)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1005..1308
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1310..1512
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1463..1483
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1715..1737
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1799..1818
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1860..1879
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1860..1878
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1879..1903
                     /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        1919..2028
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             join(2043..2045,2046..2048,2049..2759)
                     /codon_start=1
                     /product="photostable derivative of mVenus (Lee et al., 
                     2020)"
                     /label=mGold
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2088..2109)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2349..2368)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2696..2717
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2778..2954
                     /codon_start=1
                     /product="""Zdark"" protein, derived from the Z subunit of 
                     protein A, that binds to the dark state of LOV2 and is 
                     released upon irradiation (Wang et al., 2016)"
                     /label=Zdk1
                     /translation="MVDNKFNKEKTRAGAEIHSLPNLNVEQKFAFIVSLFDDPSQSANL
                     LAEAKKLNDAQAPK"
     CDS             join(2985..3170,3171..3173,3174..3566)
                     /codon_start=1
                     /product="constitutively active mutant of human Rho family 
                     small GTPase RhoA"
                     /label=RhoA (Q63L)
                     /translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGLEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     CDS             3603..3635
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             3642..3665
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3755..3774)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3820..3875
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3874..3893)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3963..4010
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4023..4728
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4111..4483
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4140..4728)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4737..4770)
                     /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(4938..5525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5017..5036)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5538..6329,6330..6398))
                     /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     6161..6180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aactgttaca ttgcaaacgt ggtttcgtgt gccaagtgtg aaaaccgatg tttaatcaag
       61 gctctgacgc atttctacaa ccacgactcc aagtgtgtgg gtgaagtcat gcatctttta
      121 atcaaatccc aagatgtgta taaaccacca aactgccaaa aaatgaaaac tgtcgacaag
      181 ctctgtccgt ttgctggcaa ctgcaagggt ctcaatccta tttgtaatta ttgaataata
      241 aaacaattat aaatgtcaaa tttgtttttt attaacgata caaaccaaac gcaacaagaa
      301 catttgtagt attatctata attgaaaacg cgtagttata atcgctgagg taatatttaa
      361 aatcattttc aaatgattca cagttaattt gcgacaatat aattttattt tcacataaac
      421 tagacgcctt gtcgtcttct tcttcgtatt ccttctcttt ttcatttttc tcttcataaa
      481 aattaacata gttattatcg tatccatata tgtatctatc gtatagagta aattttttgt
      541 tgtcataaat atatatgtct tttttaatgg ggtgtatagt accgctgcgc atagtttttc
      601 tgtaatttac aacagtgcta ttttctggta gttcttcgga gtgtgttgct ttaattatta
      661 aatttatata atcaatgaat ttgggatcgt cggttttgta caatatgttg ccggcatagt
      721 acgcagcttc ttctagttca attacaccat tttttagcag caccggatta acataacttt
      781 ccaaaatgtt gtacgaaccg ttaaacaaaa acagttcacc tcccttttct atactattgt
      841 ctgcgagcag ttgtttgttg ttaaaaataa cagccattgt aatgagacgc acaaactaat
      901 atcacaaact ggaaatgtct atcaatatat agttgctgat ggccggccta ttaatagtaa
      961 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     1021 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     1081 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     1141 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt
     1201 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac
     1261 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt
     1321 tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     1381 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     1441 cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1501 ataagcagac gtcgtttagt gaaccgtcag atcactagat gctttattgc ggtagtttat
     1561 cacagttaaa ttgctaacgc cagtctcgaa cttaacgtgc agaagttggt cgtgaggcac
     1621 tgggcaggta agtatcgggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
     1681 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     1741 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     1801 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcg gaccgaaatt
     1861 aatacgactc actatagggg aattgtgagc ggataacaat tccccggagt taatccggga
     1921 cctttaattc aacccaacac aatatattat agttaaataa gaattattat caaatcattt
     1981 gtatattaat taaaatacta tactgtaaat tacattttat ttacaatcaa aggagatata
     2041 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     2101 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     2161 acggcaagct gaccctgaag ttcatcagca ccaccggcaa gctgcccgtg ccctggccca
     2221 ccctcgtgac caccctcggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga
     2281 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2341 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2401 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2461 acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga
     2521 acggcatcaa ggcaaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg
     2581 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2641 actacctgag ctaccagtcc aaactgagca aagaccccaa cgagaagcgc gatcacatgg
     2701 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     2761 gttctggaag tggatctatg gtggataaca aattcaataa agaaaagacg cgtgccggtg
     2821 cggaaatcca ttctctgcca aacctgaatg ttgagcagaa gtttgccttc atcgtgagcc
     2881 tgtttgatga tccatctcag agcgccaatc tgctggccga agccaaaaaa ctgaacgatg
     2941 cccaggcccc aaaaggatcc ggtggttctg gttctggtgg taccatggct gccatccgga
     3001 agaaactggt gattgttggt gatggagcct gtggaaagac atgcttgctc atagtcttca
     3061 gcaaggacca gttcccagag gtgtatgtgc ccacagtgtt tgagaactat gtggcagata
     3121 tcgaggtgga tggaaagcag gtagagttgg ctttgtggga cacagctggg ctggaagatt
     3181 atgatcgcct gaggcccctc tcctacccag ataccgatgt tatactgatg tgtttttcca
     3241 tcgacagccc tgatagttta gaaaacatcc cagaaaagtg gaccccagaa gtcaagcatt
     3301 tctgtcccaa cgtgcccatc atcctggttg ggaataagaa ggatcttcgg aatgatgagc
     3361 acacaaggcg ggagctagcc aagatgaagc aggagccggt gaaacctgaa gaaggcagag
     3421 atatggcaaa caggattggc gcttttgggt acatggagtg ttcagcaaag accaaagatg
     3481 gagtgagaga ggtttttgaa atggctacga gagctgctct gcaagctaga cgtgggaaga
     3541 aaaaatctgg gtgccttgtc ttgtaaaagc ttgcggccgc acagctgtat acacgtgcaa
     3601 gccagccaga actcgccccg gaagaccccg aggatctcga gcaccaccat caccatcacc
     3661 atcactaagt gattaacctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     3721 caatgccctg gctcacaaat accactgaga tcgatctttt tccctctgcc aaaaattatg
     3781 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     3841 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     3901 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgt
     3961 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaagcat
     4021 gcggaggaaa ttctccttga agtttccctg gtgttcaaag taaaggagtt tgcaccagac
     4081 gcacctctgt tcactggtcc ggcgtattaa aacacgatac attgttatta gtacatttat
     4141 taagcgctag attctgtgcg ttgttgattt acagacaatt gttgtacgta ttttaataat
     4201 tcattaaatt tataatcttt agggtggtat gttagagcga aaatcaaatg attttcagcg
     4261 tctttatatc tgaatttaaa tattaaatcc tcaatagatt tgtaaaatag gtttcgatta
     4321 gtttcaaaca agggttgttt ttccgaaccg atggctggac tatctaatgg attttcgctc
     4381 aacgccacaa aacttgccaa atcttgtagc agcaatctag ctttgtcgat attcgtttgt
     4441 gttttgtttt gtaataaagg ttcgacgtcg ttcaaaatat tatgcgcttt tgtatttctt
     4501 tcatcactgt cgttagtgta caattgactc gacgtaaaca cgttaaatag agcttggaca
     4561 tatttaacat cgggcgtgtt agctttatta ggccgattat cgtcgtcgtc ccaaccctcg
     4621 tcgttagaag ttgcttccga agacgatttt gccatagcca cacgacgcct attaattgtg
     4681 tcggctaaca cgtccgcgat caaatttgta gttgagcttt ttggaattgc gatcgcataa
     4741 cttcgtatag catacattat acgaagttat aagctcggaa cgctgcgctc ggtcgttcgg
     4801 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4861 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4921 gccgcgttgc tggcgttttt ccataggctc cgccccctga cgagcatcac aaaaatcgac
     4981 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5041 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5101 ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg
     5161 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5221 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5281 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5341 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5401 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5461 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5521 ctcaagaaga tcctttgtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5581 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5641 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5701 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5761 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5821 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5881 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5941 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6001 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6061 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6121 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6181 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6241 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6301 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6361 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6421 tgaagcattt atcagggtta ttgtctcatg tccgcgcgtt
//
