LOCUS       Exported                7936 bp ds-DNA     circular SYN 17-AUG-2017
DEFINITION  Cys-[YPet(short)-FL-mCherry]-Cys-His(tag) tension sensor module 
            cloned into pLPCXmod plasmid.
            
            Generated for expression of fusion protein (in mamalian cells) to be
            used for force calibration measurments.
ACCESSION   .
VERSION     .
KEYWORDS    pLPCXmod + Cys-[YPet(short)-FL-mCherry]-Cys-His(tag).dna
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7936)
  AUTHORS   Anna Chrostek-Grashoff
  TITLE     Direct Submission
  JOURNAL   Exported Aug 18, 2017 from SnapGene 4.0.2
            http://www.snapgene.com
COMMENT     Cloned by inserting [EcoRI-XhoI] PCR product amplified on plasmid 
            #703 (pBSK+YPet(short)-FL-mCherry (NoStop)) into plasmid #550 
            (pLPCXmod+Cys-Ubi-[YPet(x)-Villin-mCherry]-Ubi-Cys-His(tag))
            
            
            Made by Anna Chrostek-Grashoff - 2016.04.11	
            
            MiniPreps sequenced - 2016.04.11
            MaxiPrep sequenced - 2016.04.14/18/22
            
            .
FEATURES             Location/Qualifiers
     source          1..7936
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     LTR             1..589
                     /label=5' LTR
                     /note="5' MoMuSV LTR (long terminal repeat from Moloney 
                     murine sarcoma virus)"
     misc_feature    659..1468
                     /label=-Psi(+)
                     /note="extended packaging signal"
     CDS             1565..2164
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     enhancer        2339..2642
                     /label=CMV enhancer
                     /note="human cytomegalovirus (CMV) immediate early 
                     enhancer"
     promoter        2643..2846
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2843..2867
                     /label=no.47
                     /note="pLPCX_seq_f"
     misc_feature    2912..2917
                     /label=EcoRI
                     /note="EcoRI recognition site"
     misc_feature    2921..2923
                     /note="ATG"
     misc_feature    2927..2929
                     /label=Cys
                     /note="Cystein"
     CDS             2933..3616
                     /codon_start=1
                     /product="short version of YPet 
                     
                     (yellow fluorescent protein optimized for FRET with CyPet 
                     [Nguyen and Daugherty, 2005])"
                     /label=YPet (short)
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAA"
     misc_feature    3617..3622
                     /label=BamHI \\ BglII
                     /note="sites deleted"
     CDS             3623..3868
                     /codon_start=1
                     /product="FL"
                     /note="Ferredoxin-like linker"
                     /translation="MGEFDIRFRTDDDEQFEKVLKEMNRRARKDAGTVTYTRDGNDFEI
                     RITGISEQNRKELAKEVERLAKEQNITVTYTERGSLE"
     misc_feature    3869..3874
                     /label=BamHI
                     /note="BamHI recognition site"
     CDS             3875..4582
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     misc_feature    4586..4588
                     /label=Cys
                     /note="Cystein"
     misc_feature    4589..4594
                     /label=XhoI
                     /note="XhoI recognition site"
     CDS             4595..4612
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    4613..4615
                     /note="STOP"
     misc_feature    4616..4623
                     /label=NotI
                     /note="NotI recognition site"
     primer_bind     complement(4672..4697)
                     /label=no.48
                     /note="pLPCX_seq_r"
     LTR             4675..5268
                     /label=3' LTR
                     /note="3' MoMuLV LTR (long terminal repeat from Moloney 
                     murine leukemia virus)"
     rep_origin      5937
                     /label=x
                     /note="site of replication initiation"
     rep_origin      complement(5938..6526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6697..7557)
                     /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"
ORIGIN
        1 tttgaaagac cccacccgta ggtggcaagc tagcttaagt aacgccactt tgcaaggcat
       61 ggaaaaatac ataactgaga atagaaaagt tcagatcaag gtcaggaaca aagaaacagc
      121 tgaataccaa acaggatatc tgtggtaagc ggttcctgcc ccggctcagg gccaagaaca
      181 gatgagacag ctgagtgatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg
      241 ctcggggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagtgaa
      301 tcatcagatg tttccagggt gccccaagga cctgaaaatg accctgtacc ttatttgaac
      361 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc cgctctccga gctcaataaa
      421 agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc gcccgggtac
      481 ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg ctgttccttg
      541 ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt gggggctcgt
      601 ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc
      661 agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg
      721 tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt
      781 ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg
      841 cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt
      901 aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa
      961 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct
     1021 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt
     1081 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa
     1141 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc
     1201 gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc
     1261 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt
     1321 tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc
     1381 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta
     1441 tccagccctc actccttctc taggcgccgg aatgatcccc cgggctgcag gtcggccgcc
     1501 acgaccggtg ccgccaccat cccctgaccc acgcccctga cccctcacaa ggagacgacc
     1561 ttccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc
     1621 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc
     1681 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     1741 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     1801 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     1861 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     1921 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     1981 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     2041 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     2101 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     2161 ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatggctc
     2221 cgaccgaagc cacccggggc ggccccgccg accccgcacc cgcccccgag gcccaccgac
     2281 tctagtaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     2341 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     2401 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     2461 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     2521 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     2581 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     2641 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     2701 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     2761 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     2821 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc
     2881 ggactcagat ctgggcccat aaagcttata cgaattcacc atggcttgta aaatggtgag
     2941 caaaggcgaa gagctgttca ccggcgtggt gcccatcctg gtggagctgg acggcgacgt
     3001 gaacggccac aagttcagcg tgagcggcga gggcgagggc gacgccacct acggcaagct
     3061 gaccctgaag ctgctgtgca ccaccggcaa gctgcccgtg ccctggccca ccctggtgac
     3121 caccctgggc tacggcgtgc agtgcttcgc ccggtacccc gaccacatga agcagcacga
     3181 cttcttcaag agcgccatgc ccgagggcta cgtgcaggag cggaccatct tcttcaagga
     3241 cgacggcaac tacaagaccc gggccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     3301 gatcgagctg aagggcatcg acttcaagga ggacggcaac atcctgggcc acaagctgga
     3361 gtacaactac aacagccaca acgtgtacat caccgccgac aagcagaaga acggcatcaa
     3421 ggccaacttc aagatccggc acaacatcga ggacggcggc gtgcagctgg ccgaccacta
     3481 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3541 ctaccagagc gccctgttca aggaccccaa cgagaagcgg gaccacatgg tgctgctgga
     3601 gttcctgacc gccgccggat ctatgggcga gtttgacatc cggtttcgga ctgatgacga
     3661 cgaacagttc gagaaagtgc tgaaggagat gaatcgtcga gccagaaagg atgctggaac
     3721 tgtgacctac acaagggatg ggaatgactt cgagattcgc attaccggca taagcgagca
     3781 aaaccgcaaa gaactggcca aagaggttga aaggcttgca aaggaacaga acatcacagt
     3841 cacgtatacc gagagaggtt ccctcgaagg atccatggtg agcaagggcg aggaggataa
     3901 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
     3961 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     4021 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     4081 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     4141 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     4201 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     4261 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     4321 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
     4381 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     4441 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
     4501 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
     4561 cggcatggac gagctgtaca agaaatgtct cgagcaccac caccaccacc actgagcggc
     4621 cgcctataca tcgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa
     4681 agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa
     4741 aaatacataa ctgagaatag agaagttcag atcaaggtca ggaacagatg gaacagctga
     4801 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     4861 agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg
     4921 ctcagggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa
     4981 ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact
     5041 aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa
     5101 gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc
     5161 cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg
     5221 agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcatttg ggggctcgtc
     5281 cgggatcggg agacccctgc ccagggacca ccgacccacc accgggaggt aagctggctg
     5341 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5401 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5461 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtactg
     5521 ataacttcgt ataatgtatg ctatacgaag ttattaggtc tgaagaggag tttacgtcca
     5581 gccaagctag ggccgcgatc cggaaccctt aatataactt cgtataatgt atgctatacg
     5641 aagttatcag tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     5701 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     5761 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5821 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5881 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5941 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6001 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6061 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6121 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6181 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6241 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6301 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6361 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6421 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6481 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6541 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6601 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6661 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6721 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6781 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6841 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6901 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6961 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat
     7021 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7081 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7141 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7201 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7261 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga
     7321 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7381 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7441 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7501 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7561 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7621 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7681 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     7741 cacgaggccc tttcgtcttc aagaattgct agcaattgct agcaattgct agcaattcat
     7801 accagatcac cgaaaactgt cctccaaatg tgtccccctc acactcccaa attcgcgggc
     7861 ttctgcctct tagaccactc taccctattc cccacactca ccggagccaa agccgcggcc
     7921 cttccgtttc tttgct
//
