LOCUS       sequence-357202-        7982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning plasmid for creating BcLOV4 optogenetic tool fusions. 
            Expresses 
            [BamHI]-mCherry-[XhoI]-GGGSx2-BcLOV4-[EcoRI]-GGGS-3xFLAG-STOP in a 
            pcDNA3.1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7982)
  AUTHORS   Berlew EE, Yamada K, Kuznetsov IA, Rand EA, Ochs CC, Jaber Z, 
            Gardner KH, Chow BY
  TITLE     Designing Single-Component Optogenetic Membrane Recruitment Systems:
            The Rho-Family GTPase Signaling Toolbox.
  JOURNAL   ACS Synth Biol. 2022 Jan 21;11(1):515-521. doi: 
            10.1021/acssynbio.1c00604. Epub 2022 Jan 3.
  PUBMED    34978789
REFERENCE   2  (bases 1 to 7982)
  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: 
            "10.1021/acssynbio.1c00604"; journalName: "ACS Synth Biol"; date: 
            "2022-01-21- 21"; volume: "11"; issue: "1"; pages: "515-521"
COMMENT     Sequence Label: BcLOV4 fusion tools screening plasmid #2
FEATURES             Location/Qualifiers
     source          1..7982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(58..387)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      72..207
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(126..145)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     372..392
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(401..829)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(511..532)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     723..742
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    875..1099
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1088..1105
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1140..1205)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(3036..3743)
                     /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(3311..3330)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3580..3598
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(3796..3815)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3797..3815)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3860..4063)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3889..3909)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4064..4443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4615..4634
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4709..4813
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4814..4882,4883..5674)
                     /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     complement(5032..5051)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5845..6433
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6334..6353
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6587..6604
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6721..6742
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6757..6762,6763..6780,6781..6787)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6795..6811
                     /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     6800..6822
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6819..6835
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6819..6835
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    6884..7005
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6895..6914)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6949..6968
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(7179..7973)
                     /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(7290..7309)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7900..7919
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tcctcatcct gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt
       61 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga
      121 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
      181 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
      241 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
      301 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
      361 ggactttcca caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt
      421 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
      481 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
      541 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
      601 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
      661 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
      721 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
      781 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc
      841 ctagagcccc agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca
      901 tgcctgctat tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga
      961 atagaatgac acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca
     1021 gtgggagtgg caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc
     1081 tggcaactag aaggcacagt cgaggctgat cagcgggttt aaacgggccc tctagattac
     1141 ttgtcatcgt catccttgta atcgatgtcg tggtccttat aatccccgtc atgatctttg
     1201 tagtcagatc caccaccgaa ttcaaatctt tccagtttag gagcgacagt cagcaccacc
     1261 cagccgaccc tgcccatctc atccttacag ggggtaaagt ggcacacgta cttctcctcg
     1321 gccttcttcc agcctctctc ccgattggct gtggtgccgc tgaagatgtt ctgagacttc
     1381 ttgatctcct cgatgcctgt cagcaggcct gtctccacag acacggccct gcctgctttg
     1441 atgccttccc tcaccacgtt cttaaaggtc cgtggcacag aggaagggga gtgctctgtc
     1501 agcaccttga agatatcctt gtccacgatt ggggccacca ggccattagg catattcagg
     1561 ttcagcatat ccacgatgcc ggggctgaag tacttcacga tcaggttctg ggactgtggc
     1621 tgatagctca gcaccaggta cttagaatag gcggtgtaaa actcctccac ctgtgtcttg
     1681 aactgcagct tgccgatcct atcgatcagc tcgccctcca tgccggcctc gtctctgatc
     1741 tccacggtat tggccttgta cttcttaaaa gacttaaaga aggatgtctt gccatagttg
     1801 gaggcattgt tagactcctt ggaccgcacg ctgggggtct tattagagcc ctcgatgaag
     1861 gagcctgtat cctcgtcgtt caggctcagg attctcagca cgtcggtccg gctgtggatt
     1921 gtggtagaac agttgatctg gccgcccaga aagaatctca catcgccatt gccgtccagc
     1981 agaggggaca cgtacagcag gttccaaaag ggatcgccat tcttccgata gttcagcagc
     2041 agctccactg tctcctcgcg attctcgatg gaggtgcgca gcctctttgt ggcctgccgg
     2101 tcggtaaagc tgccctgcag gaagcggcag ttccttggga tgatatcaga gcgggaatag
     2161 ccggtcacgc tcacgaatcc gtcgcttgcg aacacgatgg gattatcggc tctccgtggg
     2221 tctgtcagac agaagcaatc gcccaggcct gggtacctct ccttgtcgtt tgccagggcc
     2281 attgtggcgg atgcggtcac ctggtgcttc acaaatctag gatacaggtc agaggccagc
     2341 agcttctcga tctgctcctg ggagccattg aagatatcct ccaggccggg cagcacgttg
     2401 tgggttgctc ccttgatgtc gtggctcagg cgcctggcgt ggctattaga gatgttgatc
     2461 tgcttagggg cctcgctgct cacgtatgtg ctgtagatgg tagacagcag gcgtgtggcc
     2521 tcatcgatca gcctattgta ctcgtcgatc ttctgcagaa actccaggtt ctcggaacag
     2581 gccctgctct ggcaaaactt caggaatctg tgggctgtgg tgggattgtg cagcacgata
     2641 taaaacacct cggagctaaa gaaatcggcg aagttgccgg cagaggacag gcccaggcca
     2701 tttccgttag agccattccg gctagacacg gagccctggt tggatgggct tgtctgggtg
     2761 ctgcccttgg tggagctgcc tgcgtcgcga gagaatgtga ttctccgagg agactcggag
     2821 gtgctcttgg cggaattggg tgtgatgggg attctcttgc tgttaggggt ctgggcgcgc
     2881 tctgggcagg cgctcagagg tggcagaggg gctgtcttat tgtatgagtc gtattcgtcg
     2941 ctaaagctcc tggaaggggg gccgtctctg ttctggtagt aatggtcgat tgcgtctgtg
     3001 gccatagatc ctccacctga tccaccaccc tcgagcttgt acagctcgtc catgccgccg
     3061 gtggagtggc ggccctcggc gcgttcgtac tgttccacga tggtgtagtc ctcgttgtgg
     3121 gaggtgatgt ccaacttgat gttgacgttg taggcgccgg gcagctgcac gggcttcttg
     3181 gccttgtagg tggtcttgac ctcagcgtcg tagtggccgc cgtccttcag cttcagcctc
     3241 tgcttgatct cgcccttcag ggcgccgtcc tcggggtaca tccgctcgga ggaggcctcc
     3301 cagcccatgg tcttcttctg cattacgggg ccgtcggagg ggaagttggt gccgcgcagc
     3361 ttcaccttgt agatgaactc gccgtcctgc agggaggagt cctgggtcac ggtcaccacg
     3421 ccgccgtcct cgaagttcat cacgcgctcc cacttgaagc cctcggggaa ggacagcttc
     3481 aagtagtcgg ggatgtcggc ggggtgcttc acgtaggcct tggagccgta catgaactga
     3541 ggggacagga tgtcccaggc gaagggcagg gggccaccct tggtcacctt cagcttggcg
     3601 gtctgggtgc cctcgtaggg gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg
     3661 ttcacggagc cctccatgtg caccttgaag cgcatgaact ccttgatgat ggccatgtta
     3721 tcctcctcgc ccttgctcac catggatccg agctcggtac caagcttaag tttaaacgct
     3781 agccagcttg ggtctcccta tagtgagtcg tattaatttc gataagccag taagcagtgg
     3841 gttctctagt tagccagaga gctctgctta tatagacctc ccaccgtaca cgcctaccgc
     3901 ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt
     3961 gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa
     4021 accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga
     4081 ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat
     4141 gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca
     4201 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga
     4261 aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt
     4321 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat
     4381 atgggctatg aactaatgac cccgtaattg attactatta ataactagtc aataatcaat
     4441 gtcaacgcgt atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca
     4501 gattcttcat gcaattgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc
     4561 actactcagc gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat
     4621 cagagcagat tgtactgaga gtgcaccata ggggatcggg agatctcccg atccgtcgac
     4681 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4741 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4801 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4861 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     4921 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     4981 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5041 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5101 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5161 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5221 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5281 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5341 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5401 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5461 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5521 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5581 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5641 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5701 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5761 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5821 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5881 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5941 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6001 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6061 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6121 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6181 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6241 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6301 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6361 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6421 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6481 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6541 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     6601 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     6661 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     6721 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     6781 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     6841 ttacgccaag ctctagctag aggtcgacgg tatacagaca tgataagata cattgatgag
     6901 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     6961 gctattgctt tatttgtaac cattataagc tgcaataaac aagttggggt gggcgaagaa
     7021 ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc
     7081 gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg
     7141 cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg
     7201 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
     7261 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
     7321 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
     7381 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc
     7441 atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
     7501 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
     7561 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
     7621 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
     7681 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
     7741 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca
     7801 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
     7861 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
     7921 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac
     7981 ga
//
