LOCUS       sequence-324997-        8594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses opto-RhoGEF (BcLOV4-GGGSx2-ARHGEF11dh-GGGSx2-mCherry) in a
            pcDNA3.1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8594)
  AUTHORS   Berlew EE, Kuznetsov IA, Yamada K, Bugaj LJ, Boerckel JD, Chow BY
  TITLE     Single-Component Optogenetic Tools for Inducible RhoA GTPase 
            Signaling.
  JOURNAL   Adv Biol. 2021 Sep;5(9):e2100810. doi: 10.1002/adbi.202100810.
  PUBMED    34288599
REFERENCE   2  (bases 1 to 8594)
  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: "Adv Biol.";
            date: "2021-09"; volume: "5(9):e2100810. doi"; pages: " 
            10.1002/adbi.202100810"
COMMENT     Sequence Label: opto-RhoGEF-mCherry_pcDNA3.1
FEATURES             Location/Qualifiers
     source          1..8594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2826..3536
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2971..2989)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3239..3258
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3571..3588)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3577..3801
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3847..4275
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3934..3953)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4144..4165
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4284..4304)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4289..4618
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4469..4604
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4531..4550
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4685..5479
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4739..4758)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5349..5368
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5653..5774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5690..5709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5744..5763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5823..5839)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5823..5839)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5836..5858)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5847..5863
                     /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        complement(join(5871..5877,5878..5895,5896..5901))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5916..5937
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6054..6071)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6225..6813)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6305..6324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6984..7775,7776..7844))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFHVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7607..7626
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7845..7949)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8024..8043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8215..8594
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc
      301 ttggtaccga gctcggatcc atggccacag acgcaatcga ccattactac cagaacagag
      361 acggcccccc ttccaggagc tttagcgacg aatacgactc atacaataag acagcccctc
      421 tgccacctct gagcgcctgc ccagagcgcg cccagacccc taacagcaag agaatcccca
      481 tcacacccaa ttccgccaag agcacctccg agtctcctcg gagaatcaca ttctctcgcg
      541 acgcaggcag ctccaccaag ggcagcaccc agacaagccc atccaaccag ggctccgtgt
      601 ctagccggaa tggctctaac ggaaatggcc tgggcctgtc ctctgccggc aacttcgccg
      661 atttctttag ctccgaggtg ttttatatcg tgctgcacaa tcccaccaca gcccacagat
      721 tcctgaagtt ttgccagagc agggcctgtt ccgagaacct ggagtttctg cagaagatcg
      781 acgagtacaa taggctgatc gatgaggcca cacgcctgct gtctaccatc tacagcacat
      841 acgtgagcag cgaggcccct aagcagatca acatctctaa tagccacgcc aggcgcctga
      901 gccacgacat caagggagca acccacaacg tgctgcccgg cctggaggat atcttcaatg
      961 gctcccagga gcagatcgag aagctgctgg cctctgacct gtatcctaga tttgtgaagc
     1021 accaggtgac cgcatccgcc acaatggccc tggcaaacga caaggagagg tacccaggcc
     1081 tgggcgattg cttctgtctg acagacccac ggagagccga taatcccatc gtgttcgcaa
     1141 gcgacggatt cgtgagcgtg accggctatt cccgctctga tatcatccca aggaactgcc
     1201 gcttcctgca gggcagcttt accgaccggc aggccacaaa gaggctgcgc acctccatcg
     1261 agaatcgcga ggagacagtg gagctgctgc tgaactatcg gaagaatggc gatccctttt
     1321 ggaacctgct gtacgtgtcc cctctgctgg acggcaatgg cgatgtgaga ttctttctgg
     1381 gcggccagat caactgttct accacaatcc acagccggac cgacgtgctg agaatcctga
     1441 gcctgaacga cgaggataca ggctccttca tcgagggctc taataagacc cccagcgtgc
     1501 ggtccaagga gtctaacaat gcctccaact atggcaagac atccttcttt aagtctttta
     1561 agaagtacaa ggccaatacc gtggagatca gagacgaggc cggcatggag ggcgagctga
     1621 tcgataggat cggcaagctg cagttcaaga cacaggtgga ggagttttac accgcctatt
     1681 ctaagtacct ggtgctgagc tatcagccac agtcccagaa cctgatcgtg aagtacttca
     1741 gccccggcat cgtggatatg ctgaacctga atatgcctaa tggcctggtg gccccaatcg
     1801 tggacaagga tatcttcaag gtgctgacag agcactcccc ttcctctgtg ccacggacct
     1861 ttaagaacgt ggtgagggaa ggcatcaaag caggcagggc cgtgtctgtg gagacaggcc
     1921 tgctgacagg catcgaggag atcaagaagt ctcagaacat cttcagcggc accacagcca
     1981 atcgggagag aggctggaag aaggccgagg agaagtacgt gtgccacttt accccctgta
     2041 aggatgagat gggcagggtc ggctgggtgg tgctgactgt cgctcctaaa ctggaaagat
     2101 ttgaattcgg tggcggatca ggaggagggt caatgctgac ccagagagaa attgataggc
     2161 aggaagtgat taacgagctg tttgtgaccg aggcaagcca tctgagaacc ctgagagtgc
     2221 tggacctgat cttctaccag aggatgaaga aagagaacct gatgccccgg gaggaactgg
     2281 ctagactgtt tccaaacctg cccgagctga tcgaaattca caattcctgg tgcgaagcaa
     2341 tgaagaaact gcgcgaggaa ggacctatca ttaaggagat ttctgacctg atgctggcta
     2401 ggttcgatgg cccagcacgc gaggaactgc agcaggtggc cgctcagttt tgctcatacc
     2461 agagcatcgc cctggaactg attaagacta aacagagaaa ggagtctagg ttccagctgt
     2521 ttatgcagga ggctgaaagt cacccacagt gtcggagact gcagctgcgc gacctgatca
     2581 ttagcgaaat gcagcgactg acaaagtatc ccctgctgct ggaaagcatc atcaagcaca
     2641 ctgagggcgg gacctccgag catgaaaagc tgtgcagggc ccgcgatcag tgtcgggaga
     2701 tcctgaagta cgtgaacgag gctgtcaaac agaccgaaaa tcgacatcgg ctggagggct
     2761 atcagaaaag actggacgct acagcactgg aaagggccag tggtggtgga tcaggtggag
     2821 gatctatggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     2881 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     2941 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     3001 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     3061 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     3121 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     3181 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     3241 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     3301 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     3361 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     3421 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     3481 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaatgat
     3541 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     3601 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3661 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3721 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3781 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     3841 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3901 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3961 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4021 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4081 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4141 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4201 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4261 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     4321 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     4381 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4441 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4501 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     4561 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     4621 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     4681 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4741 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4801 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4861 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4921 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4981 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     5041 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     5101 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     5161 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     5221 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     5281 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     5341 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     5401 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     5461 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     5521 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5581 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5641 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5701 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5761 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     5821 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5881 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5941 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6001 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6061 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6121 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6181 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6241 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6301 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6361 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6421 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6481 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6541 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6601 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6661 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6721 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6781 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6841 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6901 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6961 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7021 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7081 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7141 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7201 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7261 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7321 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     7381 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7441 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7501 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7561 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7621 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7681 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7741 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7801 gcaaaaaagg gaataagggc gacatggaaa tgttgaatac tcatactctt cctttttcaa
     7861 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7921 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     7981 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     8041 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     8101 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     8161 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
     8221 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     8281 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     8341 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     8401 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     8461 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     8521 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     8581 tcgctattac catg
//
