LOCUS       Exported                7795 bp ds-DNA     circular SYN 17-AUG-2017
DEFINITION  Cys-[YPet(short)-HP35st-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.
            
            HP35st - stable Villin head peptide (st.Villin) with N27A and K29M 
            mutations (N68A, K70M in the original nomenclature).
ACCESSION   .
VERSION     .
KEYWORDS    pLPCXmod + Cys-[YPet(short)-HP35st-mCherry]-Cys-His(tag).dna
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7795)
  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 
            #423 (pLPCX+YPet(short)-st.Villin-mCherry (NoStop)) into plasmid 
            #550 (pLPCXmod+Cys-Ubi-[YPet(x)-Villin-mCherry]-Ubi-Cys-His(tag)) 
            
            Made by Anna Chrostek-Grashoff - 2014.10.30 
            
            MaxiPrep sequenced - 2014.10.30
            
            .
FEATURES             Location/Qualifiers
     source          1..7795
                     /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..3727
                     /codon_start=1
                     /product="st.Villin - ""stable Villin"" "
                     /label=HP35st
                     /note="villin head peptide with N27A and K29M mutations 
                     (N68A, K70M in the original nomenclature)"
                     /translation="LSDEDFKAVFGMTRSAFANLPLWKQQALMKEKGLF"
     misc_difference 3701..3703
                     /label=N27A
                     /note="aac -> gcc
                     
                     N68A in the HP35 nomenclature "
     misc_difference 3707..3709
                     /label=K29M
                     /note="aag -> atg
                     
                     K70M in the HP35 nomenclature "
     misc_feature    3728..3733
                     /label=BamHI
                     /note="BamHI recognition site"
     CDS             3734..4441
                     /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    4445..4447
                     /label=Cys
                     /note="Cystein"
     misc_feature    4448..4453
                     /label=XhoI
                     /note="XhoI recognition site"
     CDS             4454..4471
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    4472..4474
                     /note="STOP"
     misc_feature    4475..4482
                     /label=NotI
                     /note="NotI recognition site"
     primer_bind     complement(4531..4556)
                     /label=no.48
                     /note="pLPCX_seq_r"
     LTR             4534..5127
                     /label=3' LTR
                     /note="3' MoMuLV LTR (long terminal repeat from Moloney 
                     murine leukemia virus)"
     rep_origin      5796
                     /label=x
                     /note="site of replication initiation"
     rep_origin      complement(5797..6385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6556..7416)
                     /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 ctctgagcga tgaggacttc aaagctgtgt ttggcatgac
     3661 caggtccgca tttgccaatc ttcctctgtg gaaacaacag gccctgatga aggaaaaggg
     3721 actcttcgga tccatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     3781 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     3841 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     3901 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     3961 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     4021 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     4081 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     4141 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     4201 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     4261 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     4321 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     4381 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     4441 gaaatgtctc gagcaccacc accaccacca ctgagcggcc gcctatacat cgataaaata
     4501 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     4561 caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga
     4621 gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc
     4681 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg
     4741 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     4801 tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg
     4861 ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg
     4921 cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg
     4981 gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct
     5041 tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     5101 actacccgtc agcgggggtc tttcatttgg gggctcgtcc gggatcggga gacccctgcc
     5161 cagggaccac cgacccacca ccgggaggta agctggctgc ctcgcgcgtt tcggtgatga
     5221 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     5281 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     5341 agccatgacc cagtcacgta gcgatagcgg agtgtactga taacttcgta taatgtatgc
     5401 tatacgaagt tattaggtct gaagaggagt ttacgtccag ccaagctagg gccgcgatcc
     5461 ggaaccctta atataacttc gtataatgta tgctatacga agttatcagt actggcttaa
     5521 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     5581 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     5641 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5701 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5761 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5821 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5881 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5941 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6001 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6061 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6121 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6181 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     6241 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6301 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6361 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6421 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6481 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6541 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6601 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6661 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6721 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6781 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6841 taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt
     6901 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6961 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7021 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7081 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7141 gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag
     7201 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7261 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7321 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7381 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7441 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7501 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     7561 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca
     7621 agaattgcta gcaattgcta gcaattgcta gcaattcata ccagatcacc gaaaactgtc
     7681 ctccaaatgt gtccccctca cactcccaaa ttcgcgggct tctgcctctt agaccactct
     7741 accctattcc ccacactcac cggagccaaa gccgcggccc ttccgtttct ttgct
//
