LOCUS       sequence-99105-e        8258 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of alpha-actinin with internal stFRET inserted to measure
            mechanical stress in real time.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8258)
  AUTHORS   Meng F, Suchyna TM, Sachs F
  TITLE     A fluorescence energy transfer-based mechanical stress sensor for 
            specific proteins in situ.
  JOURNAL   FEBS J. 2008 Jun;275(12):3072-87. doi: 
            10.1111/j.1742-4658.2008.06461.x. Epub 2008 May 10.
  PUBMED    18479457
REFERENCE   2  (bases 1 to 8258)
  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: 
            "10.1111/j.1742-4658.2008.06461.x"; journalName: "FEBS J"; date: 
            "2008-06"; volume: "275"; issue: "12"; pages: "3072-87"
COMMENT     Sequence Label: Actinin-M-stFRET
FEATURES             Location/Qualifiers
     source          1..8258
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1527..1529,1530..2240)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2364..2366,2367..2369,2370..3080)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3081..3104
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     polyA_signal    5046..5167
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5083..5102)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5137..5156
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5174..5629)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5311..5332)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5523..5542
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5656..5760
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5762..6119
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5762..5953
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5785..5805)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5970..6105
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6154..6948
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             6154..6948
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6208..6227)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6818..6837
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7136..7155)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7180..7227
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7556..8144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8045..8064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatgg
      601 accattatga ttctcagcaa accaacgatt acatgcagcc agaagaggac tgggaccggg
      661 acctgctcct ggacccggcc tgggagaagc agcagagaaa gacattcacg gcatggtgta
      721 actcccacct ccggaaggcg gggacacaga tcgagaacat cgaagaggac ttccgggatg
      781 gcctgaagct catgctgctg ctggaggtca tctcaggtga acgcttggcc aagccagagc
      841 gaggcaagat gagagtgcac aagatctcca acgtcaacaa ggccctggat ttcatagcca
      901 gcaaaggcgt caaactggtg tccatcggag ccgaagaaat cgtggatggg aatgtgaaga
      961 tgaccctggg catgatctgg accatcatcc tgcgctttgc catccaggac atctccgtgg
     1021 aagagacttc agccaaggaa gggctgctcc tgtggtgtca gagaaagaca gccccttaca
     1081 aaaatgtcaa catccagaac ttccacataa gctggaagga tggcctcggc ttctgtgctt
     1141 tgatccaccg acaccggccc gagctgattg actacgggaa gctgcggaag gatgatccac
     1201 tcacaaatct gaatacggct tttgacgtgg cagagaagta cctggacatc cccaagatgc
     1261 tggatgccga agacatcgtt ggaactgccc gaccggatga gaaagccatc atgacttacg
     1321 tgtctagctt ctaccacgcc ttctctggag cccagaaggc ggagacagca gccaatcgca
     1381 tctgcaaggt gttggccgtc aaccaggaga acgagcagct tatggaagac tacgagaagc
     1441 tggccagtga tctgttggag tggatccgcc gcacaatccc gtggctggag aaccggaccg
     1501 gtggcggaaa gcttcagctg ggatccgtga gcaagggcga ggagctgttc accggggtgg
     1561 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1621 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gctgatctgc accaccggca
     1681 agctgcccgt gccctggccc accctcgtga ccaccctggg ctacggcctg cagtgcttcg
     1741 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1801 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1861 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1921 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1981 tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg
     2041 aggacggcgg cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2101 ccgtgctgct gcccgacaac cactacctga gctaccagtc caaactgagc aaagacccca
     2161 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2221 gcatggacga gctgtacaag tccggactca gatctgggct gcgcaagcgc ttacgaaaat
     2281 ttagaaacaa gattaaagaa aagcttaaaa aaattggtca gaaaatccag ggtttcgtgc
     2341 cgaaacttgc aggtgtgaat tccatggtga gcaagggcga ggagctgttc accggggtgg
     2401 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2461 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2521 agctgcccgt gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttcg
     2581 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2641 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2701 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2761 aggacggcaa catcctgggg cacaagctgg agtacaacgc catcagcgac aacgtctata
     2821 tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg
     2881 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2941 ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc aaagacccca
     3001 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     3061 gcatggacga gctgtacaag catcatcacc accatcatca ccacggaggt ggcagcggcc
     3121 gcgtgcccga gaacaccatg catgccatgc aacagaagct ggaggacttc cgggactacc
     3181 ggcgcctgca caagccgccc aaggtgcagg agaagtgcca gctggagatc aacttcaaca
     3241 cgctgcagac caagctgcgg ctcagcaacc ggcctgcctt catgccctct gagggcagga
     3301 tggtctcgga catcaacaat gcctggggct gcctggagca ggtggagaag ggctatgagg
     3361 agtggttgct gaatgagatc cggaggctgg agcgactgga ccacctggca gagaagttcc
     3421 ggcagaaggc ctccatccac gaggcctgga ctgacggcaa agaggccatg ctgcgacaga
     3481 aggactatga gaccgccacc ctctcggaga tcaaggccct gctcaagaag catgaggcct
     3541 tcgagagtga cctggctgcc caccaggacc gtgtggagca gattgccgcc atcgcacagg
     3601 agctcaatga gctggactat tatgactcac ccagtgtcaa cgcccgttgc caaaagatct
     3661 gtgaccagtg ggacaatctg ggggccctaa ctcagaagcg aagggaagct ctggagcgga
     3721 ccgagaaact gctggagacc attgaccagc tgtacttgga gtatgccaag cgggctgcac
     3781 ccttcaacaa ctggatggag ggggccatgg aggacctgca ggacaccttc attgtgcaca
     3841 ccattgagga gatccaggga ctgaccacag cccatgagca gttcaaggcc accctccctg
     3901 atgccgacaa ggagcgcctg gccatcctgg gcatccacaa tgaggtgtcc aagattgtcc
     3961 agacctacca cgtcaatatg gcgggcacca acccctacac aaccatcacg cctcaggaga
     4021 tcaatggcaa atgggaccac gtgcggcagc tggtgcctcg gagggaccaa gctctgacgg
     4081 aggagcatgc ccgacagcag cacaatgaga ggctacgcaa gcagtttgga gcccaggcca
     4141 atgtcatcgg gccctggatc cagaccaaga tggaggagat cgggaggatc tccattgaga
     4201 tgcatgggac cctggaggac cagctcagcc acctgcggca gtatgagaag agcatcgtca
     4261 actacaagcc aaagattgat cagctggagg gcgaccacca gctcatccag gaggcgctca
     4321 tcttcgacaa caagcacacc aactacacca tggagcacat ccgtgtgggc tgggagcagc
     4381 tgctcaccac catcgccagg accatcaatg aggtagagaa ccagatcctg acccgggatg
     4441 ccaagggcat cagccaggag cagatgaatg agttccgggc ctccttcaac cactttgacc
     4501 gggatcactc cggcacactg ggtcccgagg agttcaaagc ctgcctcatc agcttgggtt
     4561 atgatattgg caacgacccc cagggagaag cagaatttgc ccgcatcatg agcattgtgg
     4621 accccaaccg cctgggggta gtgacattcc aggccttcat tgacttcatg tcccgcgaga
     4681 cagccgacac agatacagca gaccaagtca tggcttcctt caagatcctg gctggggaca
     4741 agaactacat taccatggac gagctgcgcc gcgagctgcc acccgaccag gctgagtact
     4801 gcatcgcgcg gatggccccc tacaccggcc ccgactccgt gccaggtgct ctggactaca
     4861 tgtccttctc cacggcgctg tacggcgaga gtgacctcta aggtaccgcg ggcccgggat
     4921 ccaccggatc tagataactg atcataatca gccataccac atttgtagag gttttacttg
     4981 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     5041 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5101 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5161 tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     5221 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     5281 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     5341 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     5401 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     5461 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     5521 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     5581 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     5641 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5701 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5761 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     5821 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     5881 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     5941 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     6001 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     6061 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     6121 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     6181 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     6241 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     6301 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     6361 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     6421 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     6481 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     6541 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     6601 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     6661 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     6721 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     6781 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     6841 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     6901 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     6961 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     7021 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     7081 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     7141 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     7201 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     7261 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     7321 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     7381 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     7441 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7501 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7561 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7621 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7681 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     7741 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7801 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7861 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7921 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7981 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     8041 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     8101 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     8161 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     8221 cccctgattc tgtggataac cgtattaccg ccatgcat
//
