LOCUS       sequence-119521-        6353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dark mutant PA-DAD in mammalian cells, an optogenetic tool
            to activate endogenous diaphanous related formins in live cells 
            using light with LOV2 in the closed conformation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6353)
  AUTHORS   Rao MV, Chu PH, Hahn KM, Zaidel-Bar R
  TITLE     An optogenetic tool for the activation of endogenous 
            diaphanous-related formins induces thickening of stress fibers 
            without an increase in contractility.
  JOURNAL   Cytoskeleton (Hoboken). 2013 Jul;70(7):394-407. doi: 
            10.1002/cm.21115. Epub 2013 May 24.
  PUBMED    23677607
REFERENCE   2  (bases 1 to 6353)
  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"; doi: "10.1002/cm.21115"; 
            journalName: "Cytoskeleton (Hoboken)"; date: "2013-07"; volume: 
            "70"; issue: "7"; pages: "394-407"
COMMENT     Sequence Label: PA-DAD-dark
FEATURES             Location/Qualifiers
     source          1..6353
                     /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"
     CDS             join(2144..2146,2147..2857)
                     /codon_start=1
                     /product="photostable derivative of mVenus (Lee et al., 
                     2020)"
                     /label=mGold
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FISTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2186..2207)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2447..2466)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2794..2815
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2876..3301
                     /codon_start=1
                     /gene="NPH1 from Avena sativa"
                     /product="light, oxygen, or voltage domain from phototropin
                     1 of Avena sativa (Herrou and Crosson, 2011)"
                     /label=LOV2
                     /note="photoswitch that undergoes a light-inducible 
                     conformational change"
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NMRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQ
                     YFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAAKE"
     CDS             3497..3529
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             3536..3559
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3649..3668)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3713..3768
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3767..3786)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3856..3903
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3916..4621
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4004..4376
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4033..4621)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4630..4663)
                     /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(4831..5418)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4910..4929)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5431..6222,6223..6291))
                     /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     6054..6073
                     /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 ccatcacggt tctgtgagca agggcgagga
     2161 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2221 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     2281 catcagcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctcggcta
     2341 cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2401 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2461 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2521 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     2581 cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg caaacttcaa
     2641 gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc agcagaacac
     2701 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccaa
     2761 actgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2821 cgccgggatc actctcggca tggacgagct gtacaagggt tctggaagtg gatccttggc
     2881 tactacactt gaacgtattg agaagaactt tgtcattact gacccaagat tgccagataa
     2941 tcccattata ttcgcgtccg atagtttctt gcagttgaca gaatatagcc gtgaagaaat
     3001 tttgggaaga aacatgaggt ttctacaagg tcctgaaact gatcgcgcga cagtgagaaa
     3061 aattagagat gccatagata accaaacaga ggtcactgtt cagctgatta attatacaaa
     3121 gagtggtaaa aagttctgga acctctttca cttgcagcct atgcgagatc agaagggaga
     3181 tgtccagtac tttattgggg ttcagttgga tggaactgag catgtccgag atgctgccga
     3241 gagagaggga gtcatgctga ttaagaaaac tgcagaaaat atagatgagg cggcaaaaga
     3301 aggtggaagt ggtggaagtg atgagacagg tgtgatggac agtcttctag aagctctgca
     3361 gtcaggggca gcattccgac ggaagagagg gccccggcag gtcaacagga aggctgggtg
     3421 tgcagtcaca tctctgctag cctcggagct gaccaagtaa aagcttgcgg ccgcacagct
     3481 gtatacacgt gcaagccagc cagaactcgc cccggaagac cccgaggatc tcgagcacca
     3541 ccatcaccat caccatcact aagtgattaa cctcaggtgc aggctgccta tcagaaggtg
     3601 gtggctggtg tggccaatgc cctggctcac aaataccact gagatcgatc tttttccctc
     3661 tgccaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     3721 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     3781 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     3841 tatgcccata tgtaactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     3901 ttgctgaaag catgcggagg aaattctcct tgaagtttcc ctggtgttca aagtaaagga
     3961 gtttgcacca gacgcacctc tgttcactgg tccggcgtat taaaacacga tacattgtta
     4021 ttagtacatt tattaagcgc tagattctgt gcgttgttga tttacagaca attgttgtac
     4081 gtattttaat aattcattaa atttataatc tttagggtgg tatgttagag cgaaaatcaa
     4141 atgattttca gcgtctttat atctgaattt aaatattaaa tcctcaatag atttgtaaaa
     4201 taggtttcga ttagtttcaa acaagggttg tttttccgaa ccgatggctg gactatctaa
     4261 tggattttcg ctcaacgcca caaaacttgc caaatcttgt agcagcaatc tagctttgtc
     4321 gatattcgtt tgtgttttgt tttgtaataa aggttcgacg tcgttcaaaa tattatgcgc
     4381 ttttgtattt ctttcatcac tgtcgttagt gtacaattga ctcgacgtaa acacgttaaa
     4441 tagagcttgg acatatttaa catcgggcgt gttagcttta ttaggccgat tatcgtcgtc
     4501 gtcccaaccc tcgtcgttag aagttgcttc cgaagacgat tttgccatag ccacacgacg
     4561 cctattaatt gtgtcggcta acacgtccgc gatcaaattt gtagttgagc tttttggaat
     4621 tgcgatcgca taacttcgta tagcatacat tatacgaagt tataagctcg gaacgctgcg
     4681 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4741 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4801 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc tgacgagcat
     4861 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4921 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4981 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     5041 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5101 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5161 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5221 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5281 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5341 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5401 agaaaaaaag gatctcaaga agatcctttg ttaccaatgc ttaatcagtg aggcacctat
     5461 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5521 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5581 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5641 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5701 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5761 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5821 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5881 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5941 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6001 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6061 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6121 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6181 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6241 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6301 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgtccgcgc gtt
//
