LOCUS       sequence-154113-        7009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Zdk1 tagged Tiam1 Dh/PH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7009)
  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 7009)
  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-mCherry-Zdk1-TIAM1 DH/PH
FEATURES             Location/Qualifiers
     source          1..7009
                     /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             2052..2756
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2194..2212)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             2772..2948
                     /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             4152..4184
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             4191..4214
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4304..4323)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4369..4424
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4423..4442)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4512..4559
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4572..5277
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4660..5032
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4689..5277)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5286..5319)
                     /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(5487..6074)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5566..5585)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6087..6878,6879..6947))
                     /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     6710..6729
                     /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 ccatgtctag agtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     2101 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     2161 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     2221 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     2281 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     2341 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     2401 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     2461 gccctgtaat gcagaagaag actatgggct gggaggcctc ctccgagcgg atgtaccccg
     2521 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     2581 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     2641 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     2701 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagggtt
     2761 ctggaagttc catggtggat aacaaattca ataaagaaaa gacgcgtgcc ggtgcggaaa
     2821 tccattctct gccaaacctg aatgttgagc agaagtttgc cttcatcgtg agcctgtttg
     2881 atgatccatc tcagagcgcc aatctgctgg ccgaagccaa aaaactgaac gatgcccagg
     2941 ccccaaaagg atccggtggt tctggttctg gtggtaccgg cgcgccggcg accatgagac
     3001 aactctcgga tgcagataag ctgcgcaagg tgatctgcga gctcctggag acggagcgca
     3061 cctacgtgaa ggatttaaac tgtcttatgg agagatacct aaagcctctt caaaaagaaa
     3121 cttttctcac ccaggatgag cttgacgtgc tttttggaaa tttaacggaa atggtagagt
     3181 ttcaagtaga attccttaaa actctagaag atggagtgag actggtacct gatttggaaa
     3241 agcttgagaa ggttgatcaa tttaagaaag tgctgttctc tctgggggga tcattcctgt
     3301 attatgctga ccgcttcaag ctctacagtg ccttctgcgc cagccacaca aaagttccca
     3361 aggtcctggt gaaagccaag acagacacgg ctttcaaggc attcttggat gcccagaacc
     3421 cgaagcagca gcactcatcc acgctggagt cgtacctcat caagcccatc cagaggatcc
     3481 tcaagtaccc acttctgctc agggagctgt tcgccctgac cgatgcggag agcgaggagc
     3541 actaccacct ggacgtggcc atcaagacca tgaacaaggt tgccagtcac atcaatgaga
     3601 tgcagaaaat ccatgaagag tttggggctg tgtttgacca gctgattgct gaacagactg
     3661 gtgagaaaaa agaggttgca gatctgagca tgggagacct gcttttgcac actaccgtga
     3721 tctggctgaa cccgccggcc tcgctgggca agtggaaaaa ggaaccagag ttggcagcat
     3781 tcgtcttcaa aactgctgtg gtccttgtgt ataaagatgg ttccaaacag aagaagaaac
     3841 ttgtaggatc tcacaggctt tccatttatg aggactggga ccccttcaga tttcgacaca
     3901 tgatccccac ggaagcgctg caggttcgag ctttggcgag tgcagatgca gaggcaaatg
     3961 ccgtgtgtga aattgtccat gtaaaatccg agtctgaagg gaggccggag agggtctttc
     4021 acttgtgctg cagctcccca gagagccgaa aggatttcct aaaggctgtg cattcaatcc
     4081 tgcgtgataa gcacagaaga cagctcctca aaaccgagta agcggccgca cagctgtata
     4141 cacgtgcaag ccagccagaa ctcgccccgg aagaccccga ggatctcgag caccaccatc
     4201 accatcacca tcactaagtg attaacctca ggtgcaggct gcctatcaga aggtggtggc
     4261 tggtgtggcc aatgccctgg ctcacaaata ccactgagat cgatcttttt ccctctgcca
     4321 aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat
     4381 ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg
     4441 ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg
     4501 cccatatgta actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     4561 tgaaagcatg cggaggaaat tctccttgaa gtttccctgg tgttcaaagt aaaggagttt
     4621 gcaccagacg cacctctgtt cactggtccg gcgtattaaa acacgataca ttgttattag
     4681 tacatttatt aagcgctaga ttctgtgcgt tgttgattta cagacaattg ttgtacgtat
     4741 tttaataatt cattaaattt ataatcttta gggtggtatg ttagagcgaa aatcaaatga
     4801 ttttcagcgt ctttatatct gaatttaaat attaaatcct caatagattt gtaaaatagg
     4861 tttcgattag tttcaaacaa gggttgtttt tccgaaccga tggctggact atctaatgga
     4921 ttttcgctca acgccacaaa acttgccaaa tcttgtagca gcaatctagc tttgtcgata
     4981 ttcgtttgtg ttttgttttg taataaaggt tcgacgtcgt tcaaaatatt atgcgctttt
     5041 gtatttcttt catcactgtc gttagtgtac aattgactcg acgtaaacac gttaaataga
     5101 gcttggacat atttaacatc gggcgtgtta gctttattag gccgattatc gtcgtcgtcc
     5161 caaccctcgt cgttagaagt tgcttccgaa gacgattttg ccatagccac acgacgccta
     5221 ttaattgtgt cggctaacac gtccgcgatc aaatttgtag ttgagctttt tggaattgcg
     5281 atcgcataac ttcgtatagc atacattata cgaagttata agctcggaac gctgcgctcg
     5341 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5401 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5461 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gccccctgac gagcatcaca
     5521 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5581 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5641 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     5701 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5761 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5821 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5881 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5941 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6001 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6061 aaaaaggatc tcaagaagat cctttgttac caatgcttaa tcagtgaggc acctatctca
     6121 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6181 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6241 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6301 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6361 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6421 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6481 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6541 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6601 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6661 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6721 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6781 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6841 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6901 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6961 caatattatt gaagcattta tcagggttat tgtctcatgt ccgcgcgtt
//
