LOCUS       sequence-374286-        8495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EB3N-VVDfast-Halo-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 8495)
  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 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"; 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-Halo-iLID
FEATURES             Location/Qualifiers
     source          1..8495
                     /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             2106..2990
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             3090..3527
                     /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    3657..3791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3694..3713)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3748..3767
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3805..3821)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3805..3821)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3818..3840)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3829..3845
                     /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(3853..3859,3860..3877,3878..3883))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3898..3919
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4036..4053)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4207..4795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4287..4306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4966..5757,5758..5826))
                     /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     5589..5608
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5827..5931)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5999..6017
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6055..6077)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6175..6194
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6388..6410
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6402..6419
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6403..6419
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8218..8493
                     /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 gggccgccyc 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 agcttgaaat cggtactggc tttccattcg acccccatta tgtggaagtc ctgggcgagc
     2161 gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta
     2221 acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg acccatcgct
     2281 gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg ggttatttct
     2341 tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg gaagaggtcg
     2401 tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag cgcaatccag
     2461 agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc tgggacgaat
     2521 ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc ggccgcaagc
     2581 tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc
     2641 tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt gaccgcgagc
     2701 cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc
     2761 tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg ctgttctggg
     2821 gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa agcctgccta
     2881 actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac aacccggacc
     2941 tgatcggcag cgagatcgcg cgctggctgt ccacgctcga gatttccggc gctagcggag
     3001 gcggtggctc aggcggagga gggtcaggag ggggaggcag tggcgggggc ggctctggag
     3061 gagggggttc tggtggtggt agctacgtac tggcaaccac actggaacgg atcgagaaaa
     3121 atttcgtgat tactgatccg agactgcctg acaacccaat catttttgcg agcgattcct
     3181 tcctgcagct gacagaatat tctcgggaag agatcctggg gcgcaattgc cgttttctgc
     3241 agggacccga gacagaccgt gccactgttc ggaaaatcag agatgctatt gacaaccaga
     3301 ctgaagtgac cgttcagctg atcaattata ccaagagcgg caagaagttc tggaacgtgt
     3361 tccacctgca gccgatgcgc gattataagg gcgacgtcca gtacttcatt ggcgtgcagc
     3421 tggatggcac cgaacgtctt catggcgccg ctgagcgtga ggcggtctgc ctgatcaaaa
     3481 agacagcctt tcagattgct gaggcagcga acgacgaaaa ttacttttga accggtccta
     3541 gataagtaat gatcataatc agccatatca catctgtaga ggttttactt gctttaaaaa
     3601 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     3661 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3721 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3781 atgtctggat caattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct
     3841 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3901 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3961 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4021 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4081 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4141 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4201 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4261 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4321 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4381 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4441 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4501 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4561 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4621 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4681 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4741 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4801 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4861 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4921 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4981 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5041 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5101 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5161 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5221 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5281 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5341 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5401 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5461 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5521 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5581 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5641 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5701 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5761 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5821 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5881 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5941 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     6001 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     6061 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     6121 agcccgtcag ggcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     6181 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
     6241 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     6301 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     6361 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     6421 ccaagttggg atctttgcat tggcccacgg ctctcaggat ggggatgctc cccttcagca
     6481 cccggttccc cttggaaact gatggtcctg gctctgtggc atggcagtgg cactgtgagg
     6541 agccccctac cagcagcaca cagtggggtt ggcactgcca cgctccggat gccgcgctct
     6601 gatccaaccc cataatcaag ggaacccgaa ttgccccatc attgccccca ccacccccat
     6661 cctgccgggc cctcacaccc cacgctgcct tctggtgaca ttccccagcc caaacccacg
     6721 gcttcatggc taccgcgggg catttcccat tgccgcccca ttatcagctc tgcacacctc
     6781 ccgctgtacc catgcctcgt ggctgccctt ctttgacgta taatcttcta attaataccc
     6841 ggccttgtca aagtggagca caaacgttaa ttaattcccc agcaggcagg taattaacag
     6901 tgtgactccc tttttgctgc gagtggggct gatacagaga gatgtggcac tatggagccc
     6961 acggggtcct ggcactgggt gcccacggag gtccccatgt gctgcagtgt caccgcctcc
     7021 gaggagacag tattgtccct gcggtgtccc tgcagctcag ctctgtccac agggccacct
     7081 ccagtttgga ggggacacaa tgcagccccg atgcaaccca tcctcgcagc atcccaggga
     7141 caaagacccc actgcaagac cgcacacagg gctgggtccc gctcccctaa tatctacagt
     7201 gcttcttgca tggcccctta atcaatgcag ttaatcagca tgcgctcatg caccgctctg
     7261 gagctgcaaa gcccctcgca gcgctgctca ccaacaccgc gcaccgcccc ggcccagcct
     7321 gcagcacgcg ctgcaaacag gaaagaaaca aaatattgcc caaatgtagg caaaggcatt
     7381 cggctgcctt gacctccgcc gggccgggcc ctgcctgact cagctcctta ctcagcgctc
     7441 gcttcctccc tccggctgcc accgccgcag cgcacaccct gacaaagagt ggcccttaac
     7501 gggctctgag gtgcacccag cagtgcactc agcagtccaa gggccggcct ggaggtttgc
     7561 accgctacgt gctgacatta gcattgaact tggccctggg tagtgctgca ggccgggcgg
     7621 ggtgggtgta gagagtgcag cgcgcgttgc acccggtgcc ccttcccctc ccttgcatcc
     7681 cagcaggctg caccccagca ccaggcccgt gcatgcatgc tcctggtgtt attgcagcct
     7741 ggtgcatgca tgcgtcttga tggtgcagcg ctgtgcatgc atcctccttg gtgtgtagca
     7801 gtctagtgca tgcatacccc tcggtgttat tgctgctctg tgcacgcacg ctcattgtat
     7861 cacttcatcc cagtgcatgc actcacactg gagcgattgc tgctcggtgc acgcacactc
     7921 attgtatcac tgcagctcag tggctgcacg cacaccggtg ttattgctgc tcggtgcgtg
     7981 catgcacatc agtgtcgctg cagctcagtg catgcacgct cattgcccat cgctatccct
     8041 gcctctcctg ctggcgctcc ccgaggtgac ttcaagggga ccgcaggacc acctcggggg
     8101 tggggggagg gctgcacacg cggaccccgc tccccctccc caacaaagca ctgtggaatc
     8161 aaaaaggggg gaggggggat ggaggggcgc gtcacacccc cgccccacac cctcacctcg
     8221 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     8281 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggcgc
     8341 gcgccaggcg gggcggggsg gggcgrgggg sggggcgggg cgaggcggag aggtgcggcg
     8401 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     8461 cggccctata aaaagcgaag cgcgcggcgg gcggg
//
