LOCUS       sequence-374288-        8757 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SspBmicro-mCh-p60, a recruitable katanin for microtubule 
            severing, in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8757)
  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 8757)
  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: SspBmicro-mCh-p60
FEATURES             Location/Qualifiers
     source          1..8757
                     /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(1065..1271,1272..1274,1275..1394)
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein and with the mutation R74Q to reduce the 
                     affinity for iLID"
                     /label=SspB micro
                     /note="dimerizes with iLID at moderate affinity to create 
                     iLID micro (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNAQFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     CDS             1503..2207
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1645..1663)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3915..4049
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3952..3971)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4006..4025
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4063..4079)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4063..4079)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4076..4098)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4087..4103
                     /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(4111..4117,4118..4135,4136..4141))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4156..4177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4294..4311)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4465..5053)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4545..4564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5224..6015,6016..6084))
                     /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     5847..5866
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6085..6189)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6257..6275
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6313..6335)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6435..6454
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6648..6670
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6662..6679
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6663..6679
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
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 kggcggcrgg kgggggkgcc gggcggggcg
      601 gggccgccyc gggccggggg 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 catggatctg agcggcctga ccctgcagga attcagctcc ccgaaacgcc
     1081 ctaagctgct gcgtgaatat tacgattggc tggttgataa cagctttacc ccatatctgg
     1141 tggtggatgc cacatacctg ggcgtgaacg tgcccgtgga gtatgtgaaa gacggtcaga
     1201 tcgtgctgaa tctgtctgca agtgcgaccg gcaacctgca actgacaaat gattttatcc
     1261 agttcaacgc ccagtttaag ggcgtgtctc gtgaactgta tatcccgatg ggtgccgctc
     1321 tggccattta cgctcgcgag aacggcgatg gtgtgatgtt cgaaccagaa gaaatctatg
     1381 acgagctgaa tattggtcaa ttgtctagag gtggcggggg cagtggggga gggggaagtg
     1441 gaggcggcgg ttctggcggg ggaggctccg gtggcggggg gagcgggggt gggagtaagc
     1501 ttgtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     1561 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     1621 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
     1681 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
     1741 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     1801 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     1861 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggtcccgtaa
     1921 tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     1981 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
     2041 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
     2101 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     2161 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaaggct agcggaggcg
     2221 gtggctcagg cggaggaggg tcaggagggg gaggcagtgg cgggggcggc tctggaggag
     2281 ggggttctgg tggtggtagc tacgtaatga gtcttcaaat gattgttgag aatgtaaaat
     2341 tggctcgtga atatgcactg ctgggaaact atgactctgc aatggtctac tatcagggag
     2401 ttcttgacca aatgaacaag tacctgtact cagtcaaaga tacacacctc cgtcagaaat
     2461 ggcaacaggt ttggcaggaa ataaatgtgg aagctaagca agttaaggat atcatgaaaa
     2521 cactcgagag cttcaaacta gacatcactt ctttgcaagc tgcacaacat gaatttccag
     2581 ctgctgaagg agaagtctgg tccttgcctg tacctgttga gcgaagaccc ttaccaggac
     2641 ctaggaaacg ccagtcttct cagcacagtg accctaagcc acacagtaac cggccaagca
     2701 cagtcgtcag agctcatcgg ccgtctccac agaatcttca caatgacaga gggaaagctg
     2761 ttcgtagtcg tgaaaagaaa gaacagagta agggaagaga ggaaaagaac aaattacctg
     2821 ctgcagttac agaaccagag gcaaataagt ttgatggcac tgggtatgac aaggacttag
     2881 tagaagcttt ggaaagagat atcatttctc agaatcccaa cgttcggtgg tatgatattg
     2941 ctgatttagt agaagccaaa aagttgcttc aggaagcagt ggtgttacca atgtggatgc
     3001 cagaattctt taagggcatt aggagaccat ggaaaggtgt gctgatggtt ggcccacctg
     3061 gcactggaaa gacccttcta gctaaagcag ttgccacgga atgcaagaca acgttcttca
     3121 atgtctcttc atcaactctg acgtccaaat acagaggaga gtctgagaag cttgtccgtc
     3181 tcctgtttga aatggctcgg ttttactccc cagctaccat atttattgat gagatagatt
     3241 ccatttgcag ccgccgagga acttctgaag agcatgaagc cagccgaagg atgaaagctg
     3301 agctgctggt tcaaatggac ggtgttggag gtgcctcaga gaacgatgac ccttccaaaa
     3361 tggttatggt tctggcggct actaatttcc catgggacat agatgaggct ttaaggcgtc
     3421 gtctggagaa aagaatctat attccattac catcagcaaa aggcagggaa gaactgttga
     3481 gaataagtct gcgagagctg gaattggctg atgatgtcaa ccttgcgagc atagcagaaa
     3541 acatggaagg ctactcgggt gcagacatta ccaatgtatg cagggacgca tcgttgatgg
     3601 ccatgagacg gcgtattgag ggtctaaccc cagaagaaat ccggaacctt tcaagagaag
     3661 caatgcacat gcctaccacc atggaggatt ttgaaatggc tttaaagaag atttctaaat
     3721 cagtatcagc tgcagatatt gaaagatacg agaaatggat agttgagttt ggatcatgct
     3781 agacgcgtac cggtcctaga taagtaatga tcataatcag ccatatcaca tctgtagagg
     3841 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     3901 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3961 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4021 tcatcaatgt atcttatcat gtctggatca attcgtaatc atgtcatagc tgtttcctgt
     4081 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4141 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4201 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4261 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4321 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4381 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4441 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4501 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4561 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4621 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4681 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4741 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4801 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4861 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4921 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4981 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5041 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5101 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5161 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5221 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5281 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5341 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5401 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5461 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5521 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5581 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5641 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5701 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5761 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5821 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5881 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5941 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6001 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6061 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6121 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6181 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     6241 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
     6301 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     6361 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     6421 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
     6481 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct
     6541 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     6601 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     6661 ttgtaaaacg acggccagtg ccaagttggg atctttgcat tggcccacgg ctctcaggat
     6721 ggggatgctc cccttcagca cccggttccc cttggaaact gatggtcctg gctctgtggc
     6781 atggcagtgg cactgtgagg agccccctac cagcagcaca cagtggggtt ggcactgcca
     6841 cgctccggat gccgcgctct gatccaaccc cataatcaag ggaacccgaa ttgccccatc
     6901 attgccccca ccacccccat cctgccgggc cctcacaccc cacgctgcct tctggtgaca
     6961 ttccccagcc caaacccacg gcttcatggc taccgcgggg catttcccat tgccgcccca
     7021 ttatcagctc tgcacacctc ccgctgtacc catgcctcgt ggctgccctt ctttgacgta
     7081 taatcttcta attaataccc ggccttgtca aagtggagca caaacgttaa ttaattcccc
     7141 agcaggcagg taattaacag tgtgactccc tttttgctgc gagtggggct gatacagaga
     7201 gatgtggcac tatggagccc acggggtcct ggcactgggt gcccacggag gtccccatgt
     7261 gctgcagtgt caccgcctcc gaggagacag tattgtccct gcggtgtccc tgcagctcag
     7321 ctctgtccac agggccacct ccagtttgga ggggacacaa tgcagccccg atgcaaccca
     7381 tcctcgcagc atcccaggga caaagacccc actgcaagac cgcacacagg gctgggtccc
     7441 gctcccctaa tatctacagt gcttcttgca tggcccctta atcaatgcag ttaatcagca
     7501 tgcgctcatg caccgctctg gagctgcaaa gcccctcgca gcgctgctca ccaacaccgc
     7561 gcaccgcccc ggcccagcct gcagcacgcg ctgcaaacag gaaagaaaca aaatattgcc
     7621 caaatgtagg caaaggcatt cggctgcctt gacctccgcc gggccgggcc ctgcctgact
     7681 cagctcctta ctcagcgctc gcttcctccc tccggctgcc accgccgcag cgcacaccct
     7741 gacaaagagt ggcccttaac gggctctgag gtgcacccag cagtgcactc agcagtccaa
     7801 gggccggcct ggaggtttgc accgctacgt gctgacatta gcattgaact tggccctggg
     7861 tagtgctgca ggccgggcgg ggtgggtgta gagagtgcag cgcgcgttgc acccggtgcc
     7921 ccttcccctc ccttgcatcc cagcaggctg caccccagca ccaggcccgt gcatgcatgc
     7981 tcctggtgtt attgcagcct ggtgcatgca tgcgtcttga tggtgcagcg ctgtgcatgc
     8041 atcctccttg gtgtgtagca gtctagtgca tgcatacccc tcggtgttat tgctgctctg
     8101 tgcacgcacg ctcattgtat cacttcatcc cagtgcatgc actcacactg gagcgattgc
     8161 tgctcggtgc acgcacactc attgtatcac tgcagctcag tggctgcacg cacaccggtg
     8221 ttattgctgc tcggtgcgtg catgcacatc agtgtcgctg cagctcagtg catgcacgct
     8281 cattgcccat cgctatccct gcctctcctg ctggcgctcc ccgggaggtg acttcaaggg
     8341 gaccgcagga ccacctcggg ggtgggggga gggctgcaca cgcggacccc gctccccctc
     8401 cccaacaaag cactgtggaa tcaaaaaggg gggagggggg atggaggggc gcgtcacacc
     8461 cccgccccac accctcacct cgaggtgagc cccacgttct gcttcactct ccccatctcc
     8521 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
     8581 ggggcggggg ggggggggnc gcgcgccrgg sggggsgggg cggggsgrgg ggsggggsgg
     8641 ggsgaggsgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
     8701 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcggg
//
