LOCUS       sequence-319421-        5776 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  a control for optogenetic cofilin analog.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5776)
  AUTHORS   Stone OJ, Pankow N, Liu B, Sharma VP, Eddy RJ, Wang H, Putz AT, 
            Teets FD, Kuhlman B, Condeelis JS, Hahn KM
  TITLE     Optogenetic control of cofilin and alphaTAT in living cells using 
            Z-lock.
  JOURNAL   Nat Chem Biol. 2019 Dec;15(12):1183-1190. doi: 
            10.1038/s41589-019-0405-4. Epub 2019 Nov 18.
  PUBMED    31740825
REFERENCE   2  (bases 1 to 5776)
  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: 
            "10.1038/s41589-019-0405-4"; journalName: "Nat Chem Biol"; date: 
            "2019-12"; volume: "15"; issue: "12"; pages: "1183-1190"
COMMENT     Sequence Label: E. coli expression: Z-lock cofilin dark state ...
FEATURES             Location/Qualifiers
     source          1..5776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             77..99
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             118..135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             142..315
                     /codon_start=1
                     /product="""Zdark"" protein, derived from the Z subunit of 
                     protein A, that binds to the dark state of LOV2 and is 
                     released upon irradiation (Wang et al., 2016)"
                     /label=Zdk2
                     /translation="MVNKFNKEMLSARVEIYGLPNLNWGQRFAFFSSLTDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             853..1278
                     /codon_start=1
                     /gene="NPH1 from Avena sativa"
                     /product="light, oxygen, or voltage domain from phototropin
                     1 of Avena sativa (Herrou and Crosson, 2011)"
                     /label=LOV2
                     /note="photoswitch that undergoes a light-inducible 
                     conformational change"
                     /translation="ATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRN
                     ARFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQY
                     FIGVQKDGTEHVRDAAEREAVMEIKKTAEEIDEAAKEL"
     primer_bind     complement(1332..1350)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1346..1393
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1759..1764,1765..1781,1782..1787))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1814..1832)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1900..2004
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2005..2073,2074..2865)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2223..2242)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3036..3624
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3525..3544
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3778..3795
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3810..3949
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3875..3894)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4035..4057
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4051..4242)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5700..5719
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggatcgagat ctcgatcccg cgaaattaat acgactcact atagggagac cacaacggtt
       61 tccctctaga aataattttg tttaacttta agaaggagat ataccatggg cagcagccat
      121 catcatcatc atcacggtac catggttaac aaattcaata aagaaatgtt gagtgcccgt
      181 gtggaaatct atggtctgcc aaacctgaat tggggtcaga ggtttgcctt cttcagtagc
      241 ctgacggatg atccatctca gagcgccaat ctgctggccg aagccaaaaa actgaacgat
      301 gcccaggccc caaaaggttc tggaggcgga atggcctctg gtgtggctgt ctctgatgga
      361 gtcatcaagg tgttcaatga catgaaagtt cgcaagtctt caacgccaga agaagtgaag
      421 aaacgcaaga aggcagtgct cttttgcctg agtgaggaca agaagaacat catcctggag
      481 gagggcaagg agattctggt aggagatgtg gggcagactg tggacgaccc ctacaccact
      541 tttgtcaaga tgctgccaga caaggactgc cgctatgctc tctatgacgc aacctatgag
      601 accaaggaga gcaagaagga ggacctggta ttcattttct gggcccccga gagtgcaccc
      661 cttaagagca aaatgatcta tgccagctcc aaggatgcca tcaagaagaa actgacagga
      721 atcaagcacg aattacaagc taactgctac gaggaggtca aggaccgctg caccctggca
      781 gagaaactag gtggcagcgc cgtcatttcc ctggagggca agcctttggg cggtagcgga
      841 gggagcggcg gtgctactac acttgaacgt attgagaaga actttgtcat tactgaccca
      901 agattgccag ataatcccat tatattcgcg tccgatagtt tcttgcagtt gacagaatat
      961 agccgtgaag aaattttggg aagaaacgcc aggtttctac aaggtcctga aactgatcgc
     1021 gcgacagtga gaaaaattag agatgccata gataaccaaa cagaggtcac tgttcagctg
     1081 attaattata caaagagtgg taaaaagttc tggaacctct ttcacttgca gcctatgcga
     1141 gatcagaagg gagatgtcca gtactttatt ggggttcaga aggatggaac tgagcatgtc
     1201 cgagatgctg ccgagagaga ggcagtcatg gagattaaga aaactgcaga agaaattgat
     1261 gaggcggcaa aagaacttta aggatccggc tgctaacaaa gcccgaaagg aagctgagtt
     1321 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     1381 gaggggtttt ttgctgaaag gaggaactat atccggatat ccacaggacg ggtgtggtcg
     1441 ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc gagcaggact gggcggcggc
     1501 caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag aaattgcatc aacgcatata
     1561 gcgctagcag cacgccatag tgactggcga tgctgtcgga atggacgata tcccgcaaga
     1621 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
     1681 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
     1741 actgtgataa actaccgcat taaagcttat cgatgataag ctgtcaaaca tgagaattct
     1801 tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     1861 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1921 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     1981 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2041 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2101 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2161 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2221 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     2281 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2341 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2401 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2461 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     2521 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     2581 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     2641 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     2701 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     2761 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     2821 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     2881 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     2941 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3001 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3061 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3121 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3181 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3241 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3301 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3361 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3421 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3481 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3541 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3601 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3661 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3721 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3781 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3841 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3901 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3961 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4021 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4081 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     4141 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4201 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4261 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4321 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4381 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4441 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4501 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4561 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4621 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4681 cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg agatgcgccg
     4741 cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt tggtttgcgc
     4801 attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc
     4861 gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac
     4921 gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg
     4981 ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga agttaggctg
     5041 gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac
     5101 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg
     5161 aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc
     5221 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     5281 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     5341 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     5401 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     5461 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgacgctc tcccttatgc
     5521 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     5581 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     5641 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     5701 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     5761 atgcgtccgg cgtaga
//
