LOCUS       sequence-224735-       11657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NanoLuc-Firefly Luciferase fusion with constitutive nuclear 
            expression for calibration of Nanoluc signal to Firefly Luciferase 
            signal. H2B-mCherry marks chromatin with red fluorescence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11657)
  AUTHORS   Mandic A, Strebinger D, Regali C, Phillips NE, Suter DM
  TITLE     A novel method for quantitative measurements of gene expression in 
            single living cells.
  JOURNAL   Methods. 2017 May 1;120:65-75. doi: 10.1016/j.ymeth.2017.04.008. 
            Epub 2017 Apr 27.
  PUBMED    28456689
REFERENCE   2  (bases 1 to 11657)
  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"; journalName: "Methods."; 
            date: "2017-05-1"; volume: "120:65-75. doi"; pages: " 
            10.1016/j.ymeth.2017.04.008. Epub 2017 Apr 27"
COMMENT     Sequence Label: pLV-pGK-Nluc-Fluc-NLS-P2A-H2B-mCherry-IRES-Hygro
FEATURES             Location/Qualifiers
     source          1..11657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    58..175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        221..731
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     650..668
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             740..1252
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1259..2908
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     CDS             2918..2938
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2954..3010
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3017..3394
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     CDS             3401..4111
                     /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"
     primer_bind     complement(3546..3564)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3814..3833
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    join(4131..4705,4706..4708,4709..4717)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4298..4315)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4525..4544
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4718..5743
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4718..5743
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     misc_feature    5757..6345
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5810..5830)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6228..6239)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6432..6665
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6737..6858
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6774..6793)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6828..6847
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6898..7033
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7053..7072)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7054..7072)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7082..7099)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7082..7098)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7091..7113)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7240..7695
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7327..7346)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7537..7558
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7721..7825
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7826..7894,7895..8686)
                     /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     complement(8044..8063)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8857..9445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9346..9365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9599..9616
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9733..9754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9769..9774,9775..9792,9793..9799)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9807..9823
                     /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)."
     primer_bind     9812..9834
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9831..9847
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9831..9847
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9866..9886
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9868..9886
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        9914..10140
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             10141..10321
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    10368..10493
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    10986..11219
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             11404..11448
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc ggttaacttt
       61 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
      121 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttatcga
      181 tcacgagact agcctcgaga agcttgatat cgaattccac ggggttgggg ttgcgccttt
      241 tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac
      301 gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga
      361 tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg
      421 aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta
      481 gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg
      541 tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc
      601 gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat
      661 tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc
      721 tctctcccca gggggatcca tggtcttcac actcgaagat ttcgttgggg actggcgaca
      781 gacagccggc tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgtttca
      841 gaatctcggg gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg aaaatgggct
      901 gaagatcgac atccatgtca tcatcccgta tgaaggtctg agcggcgacc aaatgggcca
      961 gatcgaaaaa atttttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct
     1021 gcactatggc acactggtaa tcgacggggt tacgccgaac atgatcgact atttcggacg
     1081 gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctgtg
     1141 gaacggcaac aaaattatcg acgagcgcct gatcaacccc gacggctccc tgctgttccg
     1201 agtaaccatc aacggagtga ccggctggcg gctgtgcgaa cgcattctgg cggtcgacat
     1261 ggaagatgcc aaaaacatta agaagggccc agcgccattc tacccactcg aagacgggac
     1321 cgccggcgag cagctgcaca aagccatgaa gcgctacgcc ctggtgcccg gcaccatcgc
     1381 ctttaccgac gcacatatcg aggtggacat tacctacgcc gagtacttcg agatgagcgt
     1441 tcggctggca gaagctatga agcgctatgg gctgaataca aaccatcgga tcgtggtgtg
     1501 cagcgagaat agcttgcagt tcttcatgcc cgtgttgggt gccctgttca tcggtgtggc
     1561 tgtggcccca gctaacgaca tctacaacga gcgcgagctg ctgaacagca tgggcatcag
     1621 ccagcccacc gtcgtattcg tgagcaagaa agggctgcaa aagatcctca acgtgcaaaa
     1681 gaagctaccg atcatacaaa agatcatcat catggatagc aagaccgact accagggctt
     1741 ccaaagcatg tacaccttcg tgacttccca tttgccaccc ggcttcaacg agtacgactt
     1801 cgtgcccgag agcttcgacc gggacaaaac catcgccctg atcatgaaca gtagtggcag
     1861 taccggattg cccaagggcg tagccctacc gcaccgcacc gcttgtgtcc gattcagtca
     1921 tgcccgcgac cccatcttcg gcaaccagat catccccgac accgctatcc tcagcgtggt
     1981 gccatttcac cacggcttcg gcatgttcac cacgctgggc tacttgatct gcggctttcg
     2041 ggtcgtgctc atgtaccgct tcgaggagga gctattcttg cgcagcttgc aagactataa
     2101 gattcaatct gccctgctgg tgcccacact atttagcttc ttcgctaaga gcactctcat
     2161 cgacaagtac gacctaagca acttgcacga gatcgccagc ggcggggcgc cgctcagcaa
     2221 ggaggtaggt gaggccgtgg ccaaacgctt ccacctacca ggcatccgcc agggctacgg
     2281 cctgacagaa acaaccagcg ccattctgat cacccccgaa ggggacgaca agcctggcgc
     2341 agtaggcaag gtggtgccct tcttcgaggc taaggtggtg gacttggaca ccggtaagac
     2401 actgggtgtg aaccagcgcg gcgagctgtg cgtccgtggc cccatgatca tgagcggcta
     2461 cgttaacaac cccgaggcta caaacgctct catcgacaag gacggctggc tgcacagcgg
     2521 cgacatcgcc tactgggacg aggacgagca cttcttcatc gtggaccggc tgaagagcct
     2581 gatcaaatac aagggctacc aggtagcccc agccgaactg gagagcatcc tgctgcaaca
     2641 ccccaacatc ttcgacgccg gggtcgccgg cctgcccgac gacgatgccg gcgagctgcc
     2701 cgccgcagtc gtcgtgctgg aacacggtaa aaccatgacc gagaaggaga tcgtggacta
     2761 tgtggccagc caggttacaa ccgccaagaa gctgcgcggt ggtgttgtgt tcgtggacga
     2821 ggtgcctaaa ggactgaccg gcaagttgga cgcccgcaag atccgcgaga ttctcattaa
     2881 ggccaagaag ggcggcaaga tcgccgtgaa ttggcctcca aaaaagaaga gaaaggtaat
     2941 cgatggaagc ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa
     3001 ccctggacct tctagaatgc cagagccagc gaagtctgct cccgccccga aaaagggctc
     3061 caagaaggcg gtgactaagg cgcagaagaa agacggcaag aagcgcaagc gcagccgcaa
     3121 ggagagctat tccatctatg tgtacaaggt tctgaagcag gtccaccctg acaccggcat
     3181 ttcgtccaag gccatgggca tcatgaattc gtttgtgaac gacattttcg agcgcatcgc
     3241 aggtgaggct tcccgcctgg cgcattacaa caagcgctcg accatcacct ccagggagat
     3301 ccagacggcc gtgcgcctgc tgctgcctgg ggagttggcc aagcacgccg tgtccgaggg
     3361 tactaaggcc gtcaccaagt acaccagcgc taaggatccg atggtgagca agggcgagga
     3421 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
     3481 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     3541 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
     3601 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     3661 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     3721 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     3781 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
     3841 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     3901 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
     3961 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
     4021 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
     4081 caccggcggc atggacgagc tgtacaagta acccgggaga ataaggaccg cccctctccc
     4141 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     4201 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     4261 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     4321 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     4381 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     4441 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     4501 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     4561 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggta cacatgcttt
     4621 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     4681 ttcctttgaa aaacacgatg ataatatggc cacaaccatg aaaaagcctg aactcaccgc
     4741 gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct
     4801 ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct
     4861 gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc
     4921 atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac
     4981 ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact
     5041 gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag
     5101 ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg
     5161 tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga
     5221 caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg
     5281 ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa
     5341 tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga
     5401 ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta
     5461 cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg
     5521 cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg
     5581 ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca
     5641 aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag
     5701 tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagtaagcta gccgacaatc
     5761 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     5821 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     5881 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     5941 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     6001 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     6061 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     6121 gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct
     6181 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     6241 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     6301 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga attcgagctc
     6361 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     6421 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg
     6481 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     6541 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     6601 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     6661 tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat
     6721 atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     6781 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     6841 actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc
     6901 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     6961 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     7021 ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     7081 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     7141 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     7201 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     7261 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     7321 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     7381 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     7441 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     7501 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     7561 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     7621 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     7681 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     7741 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     7801 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     7861 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     7921 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     7981 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     8041 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     8101 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     8161 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     8221 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     8281 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     8341 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     8401 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     8461 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     8521 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     8581 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     8641 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     8701 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     8761 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     8821 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     8881 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     8941 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     9001 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     9061 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     9121 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     9181 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     9241 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     9301 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     9361 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     9421 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     9481 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     9541 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     9601 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     9661 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     9721 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     9781 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     9841 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
     9901 gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg
     9961 agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta
    10021 aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga
    10081 accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac
    10141 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
    10201 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
    10261 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
    10321 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
    10381 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
    10441 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
    10501 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
    10561 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
    10621 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
    10681 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
    10741 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
    10801 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
    10861 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
    10921 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
    10981 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
    11041 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
    11101 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
    11161 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
    11221 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
    11281 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
    11341 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
    11401 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
    11461 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
    11521 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
    11581 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
    11641 gaagaagaag gtggaga
//
