LOCUS       sequence-374287-        8331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EB3N-VVDfast-mCl3-iLID, a microtubule anchor used for 
            optogenetic recruitment of SspB-mCh-p60, in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8331)
  AUTHORS   Meiring JCM, Grigoriev I, Nijenhuis W, Kapitein LC, Akhmanova A
  TITLE     Opto-katanin, an optogenetic tool for localized, microtubule 
            disassembly.
  JOURNAL   Curr Biol. 2022 Nov 7;32(21):4660-4674.e6. doi: 
            10.1016/j.cub.2022.09.010. Epub 2022 Sep 28.
  PUBMED    36174574
REFERENCE   2  (bases 1 to 8331)
  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: 
            "10.1016/j.cub.2022.09.010"; journalName: "Curr Biol"; date: 
            "2022-11-7- 7"; volume: "32"; issue: "21"; pages: "4660-4674.e6"
COMMENT     Sequence Label: EB3N-VVDfast-mCl3-iLID
FEATURES             Location/Qualifiers
     source          1..8331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     941..963
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1026..1035
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2106..2108,2109..2111,2112..2822)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2151..2172)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2759..2780
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2922..3359
                     /codon_start=1
                     /product="improved light-inducible dimer when used with 
                     SspB nano or SspB micro (Guntas et al., 2015)"
                     /label=iLID
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNVFHLQPMRDYKGDVQ
                     YFIGVQLDGTERLHGAAEREAVCLIKKTAFQIAEAANDENYF"
     polyA_signal    3489..3623
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3526..3545)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3580..3599
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3637..3653)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3637..3653)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3650..3672)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3661..3677
                     /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(3685..3691,3692..3709,3710..3715))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3730..3751
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3868..3885)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4039..4627)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4119..4138)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4798..5589,5590..5658))
                     /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     5421..5440
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5659..5763)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5831..5849
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5887..5909)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6009..6028
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6222..6244
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6236..6253
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6237..6253
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8054..8329
                     /label=chicken beta-actin promoter
ORIGIN
        1 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
       61 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      121 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      181 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      241 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct
      301 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
      361 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
      421 gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac
      481 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tgcggggcgt
      541 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
      601 gggccgcctc gggccggggr gggctcgggg grggggcgcg gcggcccccg gagcgccggc
      661 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
      721 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
      781 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
      841 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccatc tccagcctcg gggctgtccg
      901 cagggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
      961 accggcgggg tttatatctt cccttctctg ttcctccgca gcccccaagc taggcaattg
     1021 ggcgcgccac catggccgtc aatgtgtact ccacatctgt gaccagtgaa aatctgagtc
     1081 gccatgatat gcttgcatgg gtcaacgact ccctgcacct caactatacc aagatagaac
     1141 agctttgttc aggggcagcc tactgccagt tcatggacat gctcttcccc ggctgtgtgc
     1201 acttgaggaa agtgaagttc caggccaaac tagagcatga atacatccac aacttcaagg
     1261 tgctgcaagc agctttcaag aagatgggtg ttgacaaaat cattcctgta gagaaattag
     1321 tgaaaggaaa attccaagat aattttgagt ttattcagtg gtttaagaaa ttctttgacg
     1381 caaactatga tggaaaggat tacaaccctc tgctggcgcg gcagggccag gacgtagcgc
     1441 cacctcctaa cccaggtgat cagatcttca acaaatccaa gaaactcatt ggcacagcag
     1501 ttccacagag gacgtccccc acaggcccaa aaaacatgca gacctctggc cggctgagca
     1561 atgtggcccc cccctgcatt ctccggaaga atcctccatc atctagaggt ggcgggggca
     1621 gtgggggatc ccatactctt tatgcccccg gtggatatga cattatggga tatctgtgcc
     1681 agatcatgaa ccggccaaac ccgcaggtgg aactgggccc cgtggataca tcctgcgcct
     1741 tggttctttg tgacctgaaa cagaaagaca ccccggtagt ttacgcgagt gaagccttcc
     1801 tctacatgac aggttacagc aacgcagagg tgctgggccg gaattgccgg tttctgcaaa
     1861 gccctgacgg catggtgaag cccaagagca cccggaagta cgtggatagt aacacaatca
     1921 atactatgcg caaggcaatc gacaggaatg ccgaggtgca ggttgaagta gtcaatttta
     1981 aaaagaatgg acagcgattt gttaatttcc tgactatgat acctgttagg gacgaaacag
     2041 gcgagtatcg atactctatg ggattccagt gcgaagtcga cgggagcggg ggtgggagta
     2101 agcttatggt gtctaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2161 tggacggcga cgtaaacggc cacaagttca gcgtccgcgg cgagggcgag ggcgatgcca
     2221 ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2281 ccaccctcgt gaccaccttc ggctacggcg tggcctgctt cagccgctac cccgaccaca
     2341 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2401 tctctttcaa ggacgacggt acctacaaga cccgcgccga ggtgaagttc gagggcgaca
     2461 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2521 ggcacaagct ggagtacaac ttcaacagcc actacgtcta tatcacggcc gacaagcaga
     2581 agaactgcat caaggctaac ttcaagatcc gccacaacgt tgaggacggc agcgtgcagc
     2641 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2701 accactacct gagccatcag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     2761 tggtcctgct ggagttcgtg accgccgccg ggattacaca tggcatggac gagctgtaca
     2821 aggctagcgg aggcggtggc tcaggcggag gagggtcagg agggggaggc agtggcgggg
     2881 gcggctctgg aggagggggt tctggtggtg gtagctacgt actggcaacc acactggaac
     2941 ggatcgagaa aaatttcgtg attactgatc cgagactgcc tgacaaccca atcatttttg
     3001 cgagcgattc cttcctgcag ctgacagaat attctcggga agagatcctg gggcgcaatt
     3061 gccgttttct gcagggaccc gagacagacc gtgccactgt tcggaaaatc agagatgcta
     3121 ttgacaacca gactgaagtg accgttcagc tgatcaatta taccaagagc ggcaagaagt
     3181 tctggaacgt gttccacctg cagccgatgc gcgattataa gggcgacgtc cagtacttca
     3241 ttggcgtgca gctggatggc accgaacgtc ttcatggcgc cgctgagcgt gaggcggtct
     3301 gcctgatcaa aaagacagcc tttcagattg ctgaggcagc gaacgacgaa aattactttt
     3361 gaaccggtcc tagataagta atgatcataa tcagccatat cacatctgta gaggttttac
     3421 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     3481 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3541 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3601 atgtatctta tcatgtctgg atcaattcgt aatcatgtca tagctgtttc ctgtgtgaaa
     3661 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3721 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3781 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3841 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3901 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3961 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4021 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4081 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4141 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4201 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4261 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4321 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4381 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4441 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4501 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4561 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4621 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4681 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4741 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4801 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4861 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4921 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4981 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5041 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5101 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5161 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5221 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5281 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5341 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5401 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5461 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5521 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5581 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5641 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5701 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5761 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     5821 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     5881 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     5941 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     6001 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     6061 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa
     6121 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     6181 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     6241 aacgacggcc agtgccaagt tgggatcttt gcattggccc acggctctca ggatggggat
     6301 gctccccttc agcacccggt tccccttgga aactgatggt cctggctctg tggcatggca
     6361 gtggcactgt gaggagcccc ctaccagcag cacacagtgg ggttggcact gccacgctcc
     6421 ggatgccgcg ctctgatcca accccataat caagggaacc cgaattgccc catcattgcc
     6481 cccaccaccc ccatcctgcc gggccctcac accccacgct gccttctggt gacattcccc
     6541 agcccaaacc cacggcttca tggctaccgc ggggcatttc ccattgccgc cccattatca
     6601 gctctgcaca cctcccgctg tacccatgcc tcgtggctgc ccttctttga cgtataatct
     6661 tctaattaat acccggcctt gtcaaagtgg agcacaaacg ttaattaatt ccccagcagg
     6721 caggtaatta acagtgtgac tccctttttg ctgcgagtgg ggctgataca gagagatgtg
     6781 gcactatgga gcccacgggg tcctggcact gggtgcccac ggaggtcccc atgtgctgca
     6841 gtgtcaccgc ctccgaggag acagtattgt ccctgcggtg tccctgcagc tcagctctgt
     6901 ccacagggcc acctccagtt tggaggggac acaatgcagc cccgatgcaa cccatcctcg
     6961 cagcatccca gggacaaaga ccccactgca agaccgcaca cagggctggg tcccgctccc
     7021 ctaatatcta cagtgcttct tgcatggccc cttaatcaat gcagttaatc agcatgcgct
     7081 catgcaccgc tctggagctg caaagcccct cgcagcgctg ctcaccaaca ccgcgcaccg
     7141 ccccggccca gcctgcagca cgcgctgcaa acaggaaaga aacaaaatat tgcccaaatg
     7201 taggcaaagg cattcggctg ccttgacctc cgccgggccg ggccctgcct gactcagctc
     7261 cttactcagc gctcgcttcc tccctccggc tgccaccgcc gcagcgcaca ccctgacaaa
     7321 gagtggccct taacgggctc tgaggtgcac ccagcagtgc actcagcagt ccaagggccg
     7381 gcctggaggt ttgcaccgct acgtgctgac attagcattg aacttggccc tgggtagtgc
     7441 tgcaggccgg gcggggtggg tgtagagagt gcagcgcgcg ttgcacccgg tgccccttcc
     7501 cctcccttgc atcccagcag gctgcacccc agcaccaggc ccgtgcatgc atgctcctgg
     7561 tgttattgca gcctggtgca tgcatgcgtc ttgatggtgc agcgctgtgc atgcatcctc
     7621 cttggtgtgt agcagtctag tgcatgcata cccctcggtg ttattgctgc tctgtgcacg
     7681 cacgctcatt gtatcacttc atcccagtgc atgcactcac actggagcga ttgctgctcg
     7741 gtgcacgcac actcattgta tcactgcagc tcagtggctg cacgcacacc ggtgttattg
     7801 ctgctcggtg cgtgcatgca catcagtgtc gctgcagctc agtgcatgca cgctcattgc
     7861 ccatcgctat ccctgcctct cctgctggcg ctccccggga ggtgacttca aggggaccgc
     7921 aggaccacct cgggggtggg gggagggctg cacacgcgga ccccgctccc cctccccaac
     7981 aaagcactgt ggaatcaaaa aggggggagg ggggatggag gggcgcgtca cacccccgcc
     8041 ccacaccctc acctcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     8101 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
     8161 gggggggggg gggcgcgcgc caggsggggc ggggsggggs grggggsggg gcggggcgag
     8221 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     8281 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg g
//
