LOCUS       sequence-481499-        8668 bp    DNA     circular SYN 30-JUL-2025
DEFINITION  Tagged vimentin for optogenetic vimentin pulling experiments..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8668)
  AUTHORS   Pasolli M, Meiring JCM, Conboy JP, Koenderink GH, Akhmanova A
  TITLE     Optogenetic and chemical genetic tools for rapid repositioning of 
            vimentin intermediate filaments.
  JOURNAL   J Cell Biol. 2025 Sep 1;224(9):e202504004. doi: 
            10.1083/jcb.202504004. Epub 2025 Jul 8.
  PUBMED    40627079
REFERENCE   2  (bases 1 to 8668)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2025-09-1"; volume: "224(9):e202504004. doi"; pages: 
            " 10.1083/jcb.202504004. Epub 2025 Jul 8"
COMMENT     Sequence Label: Vim-mCh-SspB
FEATURES             Location/Qualifiers
     source          1..8668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     941..963
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             2529..3233
                     /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(2671..2689)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             join(3363..3569,3570..3572,3573..3692)
                     /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"
     polyA_signal    3825..3959
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3862..3881)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3916..3935
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3973..3989)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3973..3989)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3986..4008)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3997..4013
                     /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(4021..4027,4028..4045,4046..4051))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4066..4087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4204..4221)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4375..4963)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4455..4474)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5134..5925,5926..5994))
                     /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     5757..5776
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5995..6099)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6167..6185
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6223..6245)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6345..6364
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6558..6580
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6572..6589
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6573..6589
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8390..8666
                     /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 gggccgggga gggctcgggg gaggggcgcg 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 catgtccacc aggtccgtgt cctcgtcctc ctaccgcagg atgttcggcg
     1081 gcccgggcac cgcgagccgg ccgagctcca gccggagcta cgtgactacg tccacccgca
     1141 cctacagcct gggcagcgcg ctgcgcccca gcaccagccg cagcctctac gcctcgtccc
     1201 cgggcggcgt gtatgccacg cgctcctctg ccgtgcgcct gcggagcagc gtgcccgggg
     1261 tgcggctcct gcaggactcg gtggacttct cgctggccga cgccatcaac accgagttca
     1321 agaacacccg caccaacgag aaggtggagc tacaggagct gaatgaccgc ttcgccaact
     1381 acatcgacaa ggtgcgcttc ctggagcagc agaataagat cctgctggcc gagctcgagc
     1441 agctcaaggg ccaaggcaag tcgcgcctgg gggacctcta cgaggaggag atgcgggagc
     1501 tgcgccggca ggtggaccag ctaaccaacg acaaagcccg cgtcgaggtg gagcgcgaca
     1561 acctggccga ggacatcatg cgcctccggg agaaattgca ggaggagatg cttcagagag
     1621 aggaagccga aaacaccctg caatctttca gacaggatgt tgacaatgcg tctctggcac
     1681 gtcttgacct tgaacgcaaa gtggaatctt tgcaagaaga gattgccttt ttgaagaaac
     1741 tccacgaaga ggaaatccag gagctgcagg ctcagattca ggaacagcat gtccaaatcg
     1801 atgtggatgt ttccaagcct gacctcacgg ctgccctgcg tgacgtacgt cagcaatatg
     1861 aaagtgtggc tgccaagaac ctgcaggagg cagaagaatg gtacaaatcc aagtttgctg
     1921 acctctctga ggctgccaac cggaacaatg acgccctgcg ccaggcaaag caggagtcca
     1981 ctgagtaccg gagacaggtg cagtccctca cctgtgaagt ggatgccctt aaaggaacca
     2041 atgagtccct ggaacgccag atgcgtgaaa tggaagagaa ctttgccgtt gaagctgcta
     2101 actaccaaga cactattggc cgcctgcagg atgagattca gaatatgaag gaggaaatgg
     2161 ctcgtcacct tcgtgaatac caagacctgc tcaatgttaa gatggccctt gacattgaga
     2221 ttgccaccta caggaagctg ctggaaggcg aggagagcag gatttctctg cctcttccaa
     2281 acttttcctc cctgaacctg agggaaacta atctggattc actccctctg gttgataccc
     2341 actcaaaaag gacacttctg attaagacgg ttgaaactag agatggacag gttatcaacg
     2401 aaacttctca gcatcacgat gaccttgaat ctagaggtgg cgggggcagt gggggagggg
     2461 gaagtggagg cggcggttct ggcgggggag gctccggtgg cggggggagc ggggggggga
     2521 gtaagcttgt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     2581 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     2641 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     2701 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     2761 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     2821 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     2881 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggtc
     2941 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     3001 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     3061 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     3121 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     3181 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aaggctagcg
     3241 gaggcggtgg ctcaggcgga ggagggtcag gagggggagg cagtggcggg ggcggctctg
     3301 gaggaggggg ttctggtggt ggtagctacg tagatctgag cggcctgacc ctgcaggaat
     3361 tcagctcccc gaaacgccct aagctgctgc gtgaatatta cgattggctg gttgataaca
     3421 gctttacccc atatctggtg gtggatgcca catacctggg cgtgaacgtg cccgtggagt
     3481 atgtgaaaga cggtcagatc gtgctgaatc tgtctgcaag tgcgaccggc aacctgcaac
     3541 tgacaaatga ttttatccag ttcaacgccc agtttaaggg cgtgtctcgt gaactgtata
     3601 tcccgatggg tgccgctctg gccatttacg ctcgcgagaa cggcgatggt gtgatgttcg
     3661 aaccagaaga aatctatgac gagctgaata ttggttaaac cggtcctaga taagtaatga
     3721 tcataatcag ccatatcaca tctgtagagg ttttacttgc tttaaaaaac ctcccacacc
     3781 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     3841 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3901 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatca
     3961 attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4021 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4081 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4141 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4201 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4261 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4321 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4381 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4441 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4501 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4561 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4621 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4681 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4741 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4801 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4861 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4921 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4981 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5041 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5101 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5161 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5221 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5281 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5341 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5401 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5461 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5521 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5581 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5641 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5701 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5761 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5821 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5881 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5941 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6001 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6061 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6121 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     6181 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     6241 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     6301 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     6361 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     6421 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     6481 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     6541 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg ccaagttggg
     6601 atctttgcat tggcccacgg ctctcaggat ggggatgctc cccttcagca cccggttccc
     6661 cttggaaact gatggtcctg gctctgtggc atggcagtgg cactgtgagg agccccctac
     6721 cagcagcaca cagtggggtt ggcactgcca cgctccggat gccgcgctct gatccaaccc
     6781 cataatcaag ggaacccgaa ttgccccatc attgccccca ccacccccat cctgccgggc
     6841 cctcacaccc cacgctgcct tctggtgaca ttccccagcc caaacccacg gcttcatggc
     6901 taccgcgggg catttcccat tgccgcccca ttatcagctc tgcacacctc ccgctgtacc
     6961 catgcctcgt ggctgccctt ctttgacgta taatcttcta attaataccc ggccttgtca
     7021 aagtggagca caaacgttaa ttaattcccc agcaggcagg taattaacag tgtgactccc
     7081 tttttgctgc gagtggggct gatacagaga gatgtggcac tatggagccc acggggtcct
     7141 ggcactgggt gcccacggag gtccccatgt gctgcagtgt caccgcctcc gaggagacag
     7201 tattgtccct gcggtgtccc tgcagctcag ctctgtccac agggccacct ccagtttgga
     7261 ggggacacaa tgcagccccg atgcaaccca tcctcgcagc atcccaggga caaagacccc
     7321 actgcaagac cgcacacagg gctgggtccc gctcccctaa tatctacagt gcttcttgca
     7381 tggcccctta atcaatgcag ttaatcagca tgcgctcatg caccgctctg gagctgcaaa
     7441 gcccctcgca gcgctgctca ccaacaccgc gcaccgcccc ggcccagcct gcagcacgcg
     7501 ctgcaaacag gaaagaaaca aaatattgcc caaatgtagg caaaggcatt cggctgcctt
     7561 gacctccgcc gggccgggcc ctgcctgact cagctcctta ctcagcgctc gcttcctccc
     7621 tccggctgcc accgccgcag cgcacaccct gacaaagagt ggcccttaac gggctctgag
     7681 gtgcacccag cagtgcactc agcagtccaa gggccggcct ggaggtttgc accgctacgt
     7741 gctgacatta gcattgaact tggccctggg tagtgctgca ggccgggcgg ggtgggtgta
     7801 gagagtgcag cgcgcgttgc acccggtgcc ccttcccctc ccttgcatcc cagcaggctg
     7861 caccccagca ccaggcccgt gcatgcatgc tcctggtgtt attgcagcct ggtgcatgca
     7921 tgcgtcttga tggtgcagcg ctgtgcatgc atcctccttg gtgtgtagca gtctagtgca
     7981 tgcatacccc tcggtgttat tgctgctctg tgcacgcacg ctcattgtat cacttcatcc
     8041 cagtgcatgc actcacactg gagcgattgc tgctcggtgc acgcacactc attgtatcac
     8101 tgcagctcag tggctgcacg cacaccggtg ttattgctgc tcggtgcgtg catgcacatc
     8161 agtgtcgctg cagctcagtg catgcacgct cattgcccat cgctatccct gcctctcctg
     8221 ctggcgctcc ccgggaggtg acttcaaggg gaccgcagga ccacctcggg ggtgggggga
     8281 gggctgcaca cgcggacccc gctccccctc cccaacaaag cactgtggaa tcaaaaaggg
     8341 gggagggggg atggaggggc gcgtcacacc cccgccccac accctcacct cgaggtgagc
     8401 cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta
     8461 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggc gcgcgccagg
     8521 gggggggggg gggggggggg ggggggggcg gggcgaggcg gagaggtgcg gcggcagcca
     8581 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
     8641 ataaaaagcg aagcgcgcgg cgggcggg
//
