LOCUS       sequence-324999-        8495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses opto-RhoA (RhoA-GGGSx2-BcLOV4-GGGSx2-mCherry) in a 
            pcDNA3.1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8495)
  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 8495)
  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-RhoA-mCherry_pcDNA3.1
FEATURES             Location/Qualifiers
     source          1..8495
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     213..233
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(242..670)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(352..373)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     564..583
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    716..940
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     929..946
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(981..1691)
                     /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(1259..1278)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1528..1546
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4150..4169)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4151..4169)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4214..4417)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4243..4263)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4418..4797
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4969..4988
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5063..5167
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5168..5236,5237..6028)
                     /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     complement(5386..5405)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6199..6787
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6688..6707
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6941..6958
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7075..7096
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7111..7116,7117..7134,7135..7141)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7149..7165
                     /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)."
     primer_bind     7154..7176
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7173..7189
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7173..7189
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    7238..7359
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7249..7268)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7303..7322
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(7533..8327)
                     /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(7644..7663)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     8254..8273
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(8394..8495,1..228))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(8408..8495,1..48)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8462..8481)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag
       61 gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
      121 ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact
      181 tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagaat
      241 taattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
      301 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
      361 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
      421 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
      481 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
      541 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
      601 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
      661 cagggcgcgt ggggataccc cctagagccc cagctggttc tttccgcctc agaagccata
      721 gagcccaccg catccccagc atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc
      781 tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc
      841 tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg
      901 gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcgaggctga tcagcgggtt
      961 taaacgggcc ctctagatca ttacttgtac agctcgtcca tgccgccggt ggagtggcgg
     1021 ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc
     1081 aacttgatgt tgacgttgta ggcgccgggc agctgcacgg gcttcttggc cttgtaggtg
     1141 gtcttgacct cagcgtcgta gtggccgccg tccttcagct tcagcctctg cttgatctcg
     1201 cccttcaggg cgccgtcctc ggggtacatc cgctcggagg aggcctccca gcccatggtc
     1261 ttcttctgca ttacggggcc gtcggagggg aagttggtgc cgcgcagctt caccttgtag
     1321 atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg
     1381 aagttcatca cgcgctccca cttgaagccc tcggggaagg acagcttcaa gtagtcgggg
     1441 atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca tgaactgagg ggacaggatg
     1501 tcccaggcga agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc
     1561 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc
     1621 tccatgtgca ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc
     1681 ttgctcacca tagatcctcc acctgatcca ccaccgaatt caaatctttc cagtttagga
     1741 gcgacagtca gcaccaccca gccgaccctg cccatctcat ccttacaggg ggtaaagtgg
     1801 cacacgtact tctcctcggc cttcttccag cctctctccc gattggctgt ggtgccgctg
     1861 aagatgttct gagacttctt gatctcctcg atgcctgtca gcaggcctgt ctccacagac
     1921 acggccctgc ctgctttgat gccttccctc accacgttct taaaggtccg tggcacagag
     1981 gaaggggagt gctctgtcag caccttgaag atatccttgt ccacgattgg ggccaccagg
     2041 ccattaggca tattcaggtt cagcatatcc acgatgccgg ggctgaagta cttcacgatc
     2101 aggttctggg actgtggctg atagctcagc accaggtact tagaataggc ggtgtaaaac
     2161 tcctccacct gtgtcttgaa ctgcagcttg ccgatcctat cgatcagctc gccctccatg
     2221 ccggcctcgt ctctgatctc cacggtattg gccttgtact tcttaaaaga cttaaagaag
     2281 gatgtcttgc catagttgga ggcattgtta gactccttgg accgcacgct gggggtctta
     2341 ttagagccct cgatgaagga gcctgtatcc tcgtcgttca ggctcaggat tctcagcacg
     2401 tcggtccggc tgtggattgt ggtagaacag ttgatctggc cgcccagaaa gaatctcaca
     2461 tcgccattgc cgtccagcag aggggacacg tacagcaggt tccaaaaggg atcgccattc
     2521 ttccgatagt tcagcagcag ctccactgtc tcctcgcgat tctcgatgga ggtgcgcagc
     2581 ctctttgtgg cctgccggtc ggtaaagctg ccctgcagga agcggcagtt ccttgggatg
     2641 atatcagagc gggaatagcc ggtcacgctc acgaatccgt cgcttgcgaa cacgatggga
     2701 ttatcggctc tccgtgggtc tgtcagacag aagcaatcgc ccaggcctgg gtacctctcc
     2761 ttgtcgtttg ccagggccat tgtggcggat gcggtcacct ggtgcttcac aaatctagga
     2821 tacaggtcag aggccagcag cttctcgatc tgctcctggg agccattgaa gatatcctcc
     2881 aggccgggca gcacgttgtg ggttgctccc ttgatgtcgt ggctcaggcg cctggcgtgg
     2941 ctattagaga tgttgatctg cttaggggcc tcgctgctca cgtatgtgct gtagatggta
     3001 gacagcaggc gtgtggcctc atcgatcagc ctattgtact cgtcgatctt ctgcagaaac
     3061 tccaggttct cggaacaggc cctgctctgg caaaacttca ggaatctgtg ggctgtggtg
     3121 ggattgtgca gcacgatata aaacacctcg gagctaaaga aatcggcgaa gttgccggca
     3181 gaggacaggc ccaggccatt tccgttagag ccattccggc tagacacgga gccctggttg
     3241 gatgggcttg tctgggtgct gcccttggtg gagctgcctg cgtcgcgaga gaatgtgatt
     3301 ctccgaggag actcggaggt gctcttggcg gaattgggtg tgatggggat tctcttgctg
     3361 ttaggggtct gggcgcgctc tgggcaggcg ctcagaggtg gcagaggggc tgtcttattg
     3421 tatgagtcgt attcgtcgct aaagctcctg gaaggggggc cgtctctgtt ctggtagtaa
     3481 tggtcgattg cgtctgtggc cattgaccct cctcctgatc cgccaccccc agattttttc
     3541 ttcccacgtc tagcttgcag agcagctctc gtagccattt caaaaacctc tctcactcca
     3601 tctttggtct ttgctgaaca ctccatgtac ccaaaagcgc caatcctgtt tgccatatct
     3661 ctgccttctt caggtttcac cggctcctgc ttcatcttgg ctagctcccg ccttgtgtgc
     3721 tcatcattcc gaagatcctt cttattccca accaggatga tgggcacgtt gggacagaaa
     3781 tgcttgactt ctggggtcca cttttctggg atgttttcta aactatcagg gctgtcgatg
     3841 gaaaaacaca tcagtataac atcggtatct gggtaggaga ggggcctcag gcggtcataa
     3901 tcttcctgcc cagctgtgtc ccacaaagcc aactctacct gctttccatc cacctcgata
     3961 tctgccacat agttctcaaa cactgtgggc acatacacct ctgggaactg gtccttgctg
     4021 aagactatga gcaagcatgt ctttccacag gctccatcac caacaatcac cagtttcttc
     4081 cggatggcag ctgacatgga tccgagctcg gtaccaagct taagtttaaa cgctagccag
     4141 cttgggtctc cctatagtga gtcgtattaa tttcgataag ccagtaagca gtgggttctc
     4201 tagttagcca gagagctctg cttatataga cctcccaccg tacacgccta ccgcccattt
     4261 gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa
     4321 acaaactccc attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct
     4381 atccacgccc attgatgtac tgccaaaacc gcatcaccat ggtaatagcg atgactaata
     4441 cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg
     4501 cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat
     4561 gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc
     4621 ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg
     4681 gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc
     4741 tatgaactaa tgaccccgta attgattact attaataact agtcaataat caatgtcaac
     4801 gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg cctaacccta agcagattct
     4861 tcatgcaatt gtcggtcaag ccttgccttg ttgtagctta aattttgctc gcgcactact
     4921 cagcgacctc caacacacaa gcagggagca gatactggct taactatgcg gcatcagagc
     4981 agattgtact gagagtgcac cataggggat cgggagatct cccgatccgt cgacgtcagg
     5041 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5101 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5161 gaagagtatg agtattcaac atttccatgt cgcccttatt cccttttttg cggcattttg
     5221 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5281 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5341 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5401 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5461 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5521 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5581 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5641 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5701 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5761 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5821 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5881 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5941 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6001 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6061 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6121 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6181 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6241 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6301 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6361 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6421 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6481 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6541 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6601 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6661 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6721 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6781 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6841 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     6901 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6961 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     7021 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     7081 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     7141 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     7201 caagctctag ctagaggtcg acggtataca gacatgataa gatacattga tgagtttgga
     7261 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     7321 gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga agaactccag
     7381 catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc
     7441 caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc
     7501 ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag
     7561 aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc
     7621 cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg
     7681 tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg
     7741 atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgcgc
     7801 gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca
     7861 tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct
     7921 tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc
     7981 atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact
     8041 tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa
     8101 ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg
     8161 gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac
     8221 acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc
     8281 acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct
     8341 catcctgtct cttgatcaga tccgaaaatg gatatacaag ctcccgggag ctttttgcaa
     8401 aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggcagaggcg
     8461 gcctcggcct ctgcataaat aaaaaaaatt agtca
//
