LOCUS       sequence-154111-        6457 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Zdk1 tagged Rac1 Q61L.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6457)
  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 6457)
  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-Rac1 Q61L
FEATURES             Location/Qualifiers
     source          1..6457
                     /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..3164,3165..3167,3168..3563)
                     /codon_start=1
                     /product="constitutively active mutant of human Rac family 
                     small GTPase 1"
                     /label=Rac1 (Q61L)
                     /translation="MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGLEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     CDS             3600..3632
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             3639..3662
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3752..3771)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3817..3872
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3871..3890)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3960..4007
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4020..4725
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4108..4480
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4137..4725)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4734..4767)
                     /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(4935..5522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5014..5033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5535..6326,6327..6395))
                     /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     6158..6177
                     /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 taccatgcag gccatcaagt
     3001 gtgtggtggt gggagacgga gctgtaggta aaacttgcct actgatcagt tacacaacca
     3061 atgcatttcc tggagaatat atccctactg tctttgacaa ttattctgcc aatgttatgg
     3121 tagatggaaa accggtgaat ctgggcttat gggatacagc tggactagaa gattatgaca
     3181 gattacgccc cctatcctat ccgcaaacag atgtgttctt aatttgcttt tcccttgtga
     3241 gtcctgcatc atttgaaaat gtccgtgcaa agtggtatcc tgaggtgcgg caccactgtc
     3301 ccaacactcc catcatccta gtgggaacta aacttgatct tagggatgat aaagacacga
     3361 tcgagaaact gaaggagaag aagctgactc ccatcaccta tccgcagggt ctagccatgg
     3421 ctaaggagat tggtgccgta aaatacctgg agtgctcggc gctcacacag cgaggcctca
     3481 agacagtgtt tgacgaagcg atccgagcag tcctctgccc gcctcccgtg aagaagagga
     3541 agagaaaatg cctgctgttg taaaagcttg cggccgcaca gctgtataca cgtgcaagcc
     3601 agccagaact cgccccggaa gaccccgagg atctcgagca ccaccatcac catcaccatc
     3661 actaagtgat taacctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa
     3721 tgccctggct cacaaatacc actgagatcg atctttttcc ctctgccaaa aattatgggg
     3781 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     3841 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     3901 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgtaac
     3961 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaagcatgcg
     4021 gaggaaattc tccttgaagt ttccctggtg ttcaaagtaa aggagtttgc accagacgca
     4081 cctctgttca ctggtccggc gtattaaaac acgatacatt gttattagta catttattaa
     4141 gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt gtacgtattt taataattca
     4201 ttaaatttat aatctttagg gtggtatgtt agagcgaaaa tcaaatgatt ttcagcgtct
     4261 ttatatctga atttaaatat taaatcctca atagatttgt aaaataggtt tcgattagtt
     4321 tcaaacaagg gttgtttttc cgaaccgatg gctggactat ctaatggatt ttcgctcaac
     4381 gccacaaaac ttgccaaatc ttgtagcagc aatctagctt tgtcgatatt cgtttgtgtt
     4441 ttgttttgta ataaaggttc gacgtcgttc aaaatattat gcgcttttgt atttctttca
     4501 tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt taaatagagc ttggacatat
     4561 ttaacatcgg gcgtgttagc tttattaggc cgattatcgt cgtcgtccca accctcgtcg
     4621 ttagaagttg cttccgaaga cgattttgcc atagccacac gacgcctatt aattgtgtcg
     4681 gctaacacgt ccgcgatcaa atttgtagtt gagctttttg gaattgcgat cgcataactt
     4741 cgtatagcat acattatacg aagttataag ctcggaacgc tgcgctcggt cgttcggctg
     4801 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4861 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4921 gcgttgctgg cgtttttcca taggctccgc cccctgacga gcatcacaaa aatcgacgct
     4981 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5041 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5101 tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt
     5161 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5221 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5281 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5341 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5401 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5461 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5521 aagaagatcc tttgttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5581 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5641 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5701 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5761 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5821 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5881 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5941 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6001 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6061 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6121 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6181 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6241 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6301 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6361 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6421 agcatttatc agggttattg tctcatgtcc gcgcgtt
//
