LOCUS       pTL50-EMMA-PUFFF        9298 bp DNA     circular SYN 18-JUL-2023
DEFINITION  pTL5 PUFFFIN random integration plasmid to create PUFFFIN secretors 
            with nuclear, red fluorescent mCherry-3NLS that remains inside the 
            secretors and with secreted, green fluorescent s36GFP-mNG that is 
            transferred to nearby cells resulting in neighbour labelling. 
            Correct secretors can be isolated by fluorescence activated cell 
            sorting screening for high levels of green and red fluorescence 
            and/or selected by treatment with puromycin antibiotic. 
             
            The PUFFFIN cassette was assembled via EMMA cloning [1] in the EMMA 
            toolkit receiver vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9298)
  AUTHORS   Martella A, Matjusaitis M, Auxillos J, Pollard SM, Cai Y
  TITLE     EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid
            Design and Production of Diverse Expression Vectors.
  JOURNAL   ACS Synth Biol. 2017 Apr 24. doi: 10.1021/acssynbio.7b00016.
  PUBMED    28418644
REFERENCE   2  (bases 1 to 9298)
  AUTHORS   Tamina Lebek
  TITLE     Direct Submission
  JOURNAL   Exported Jul 2, 2024 from SnapGene 7.2.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2017 Apr 24. doi: 10.1021/acssynbio.7b00016."
FEATURES             Location/Qualifiers
     source          1..9298
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    37..40
                     /label=pV/1-connector
     misc_feature    52..1265
                     /locus_tag="cHS4 Chicken B-globin insulator"
                     /label=cHS4 Chicken B-globin insulator
                     /note="cHS4 insulators are flanking the PUFFFIN cassette to
                     prevent gene silencing at the random integration site. This
                     will ensure stable PUFFFIN expression even when cells are 
                     cultured for a long time."
     misc_feature    1266..1269
                     /label=p3/4-connector
     promoter        1331..2985
                     /locus_tag="CAG"
                     /label=CAG
                     /note="The CAG promoter is the strongest, ubiquitous 
                     promoter we have in the lab and should lead to high 
                     expression levels of the PUFFFIN cassette."
     enhancer        1331..1636
                     /locus_tag="CMV enhancer"
                     /label=CMV enhancer
     primer_bind     complement(1342..1361)
                     /label=MMS140_CAG-50R
     primer_bind     complement(1538..1554)
                     /label=MMS140_CAG-50R
     promoter        1637..1913
                     /locus_tag="Chiken beta Actin promoter"
                     /label=Chiken beta Actin promoter
     promoter        1915..2985
                     /locus_tag="Rabbit beta globin splice acceptor"
                     /label=Rabbit beta globin splice acceptor
     promoter        1915..2985
                     /locus_tag="CAG promoter"
                     /label=CAG promoter
     primer_bind     2892..2913
                     /label=TLS1_CAG_F_58C
     primer_bind     2937..2947
                     /label=TLS3_36GFP_F_62C
     primer_bind     2937..2947
                     /label=TLS3_36GFP_F_62Ca
     primer_bind     2937..2947
                     /label=TLS3_36GFP_F_62C`b
     primer_bind     2958..3033
                     /label=TLP7_OEP_F_68
     primer_bind     2958..3027
                     /label=TLP5_OEP_F_64
     primer_bind     2992..3015
                     /label=TLP8_4/5-AscI_F_68
     primer_bind     2992..3015
                     /label=TLP8_4/5-AscI_F_68a
     misc_feature    2992..2995
                     /label=p4/5-connector
     primer_bind     2997..3015
                     /label=TLP3_XhoI_F_64C
     primer_bind     3006..3034
                     /label=TLP1_s36GFP_F_72C
     misc_feature    3016..3024
                     /label=kozak
     CDS             join(3025..3078,3079..3084,3085..3804,3805..3846,
                     3847..4551)
                     /codon_start=1
                     /product="Secretion signal of human serum albumin precursor
                     for strong secretion. Optimised for expression in mammalian
                     cells: balanced for GC content 53% (instead 42%), and 
                     improved 0.96 CAI (codon adaptation index, instead of 0.65 
                     in original sequence). Original sequence see Dugaiczyk et 
                     al. PNAS 79, 71–75 (1982)."
                     /product="The short 2AA RG linker maintains sequence 
                     context of HSA precursor to prevent issues with cleavage of
                     the secretion signal upon secretion."
                     /product="+36GFP sequence optimised for expression in 
                     mammalian cells see Bar-Shir et al. Chem Commun (Camb) 51, 
                     4869–4871 (2015). +36GFP was chosen because it was shown to
                     be easily taken up by mammalian cells when exogenously 
                     introduced see McNaughton et al. PNAS 106,6111–6116 (2009) 
                     and Cronican et al. ACS Chem. Biol. 5, 747–752 (2010) 
                     (where it performed better than other supercharged GFP 
                     versions and better than e.g.,Tat-mCherry which was used 
                     for the design of the Cherry niche by Ombrato et al. Nature
                     572, 603–608 (2019). +36GFP with the N-terminal secretion 
                     signal and the RG linker is referred to as s36GFP."
                     /product="14-linker. Glycine-serine linkers are commonly 
                     used in immunology whereas the small polar amino acid 
                     threonine introduces flexibility while maintaining 
                     stability of the linker as described by Chen et al. 
                     Advanced Drug Delivery Reviews 65, 1357–1369 (2013). This 
                     makes the 14 linker a short, stable, and flexible linker 
                     that will keep s36GFP and mNeonGreen close together to 
                     avoid problems with secretion while still allowing the 
                     flexibility necessary with the high positive charge of 
                     s36GFP."
                     /product="mNeonGreen (mNG) with stop codon, DNA sequence 
                     see Shaner et al. Nat Methods 10, 407–409 (2013). mNG was 
                     chosen as one of the brightest green fluorescent proteins 
                     to increase the green fluorescence signal in flow cytometry
                     for clear distinction of unlabelled and labelled receivers 
                     and for live imaging as s36GFP appeared not to be bright 
                     enough. +36GFP will be able to drag mNG through the cell 
                     membrane to enable uptake as shown for mCherry by Cronican 
                     et al. ACS Chem. Biol. 5, 747–752 (2010)."
                     /label=s36GFP-mNG tandem construct
                     /translation="MKWVTFISLLFLFSSAYSRGGGASKGERLFRGKVPILVELKGDVN
                     GHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHD
                     FFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKL
                     RYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHY
                     LSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKGGSGGTSGGGGGSGVSKGEED
                     NMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHI
                     GYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQ
                     VKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTY
                     TFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK"
     primer_bind     3354..3370
                     /label=TLS3_36GFP_F_62C
     primer_bind     3354..3370
                     /label=TLS3_36GFP_F_62Ca
     primer_bind     3354..3370
                     /label=TLS3_36GFP_F_62C`b
     primer_bind     complement(3453..3473)
                     /label=TLS4_36GFP_R_61C
     primer_bind     complement(3820..3847)
                     /label=TLP48_OEP_14-HT_R_83
     primer_bind     3836..3862
                     /label=TLP12_p6/7-mCherry_F_73
     primer_bind     complement(3839..3853)
                     /label=OEP_14_R
     primer_bind     3847..3862
                     /label=TLP21_p13/14-mCherry_F_63
     primer_bind     3934..3953
                     /label=TLS7_mNG_F_63C
     primer_bind     complement(4025..4046)
                     /label=TLS8_mNG_R_55C
     primer_bind     complement(4530..4556)
                     /label=TLP47_p10/11-mNG_NoStop_R_61
     primer_bind     complement(4530..4551)
                     /label=TLP33_p10/11-mNG_R_63
     primer_bind     complement(4539..4551)
                     /label=TLP9_p5/6-NotI_R_66
     primer_bind     complement(4539..4551)
                     /label=TLP9_p5/6-NotI_R_66a
     misc_feature    4552..4555
                     /label=p10/11 connector
     CDS             4558..4611
                     /locus_tag="T2A"
                     /label=T2A
     primer_bind     4613..4644
                     /label=TLP19_p11/12-PAC_F_71
     misc_feature    4614..4617
                     /label=p11/12-connector
     CDS             4618..5215
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4737..4757)
                     /label=TLS11_PAC_R_67C
     misc_feature    5004..5009
                     /locus_tag="Mutated BsmBI"
                     /label=Mutated BsmBI
                     /note="/ApEinfo_fwdcolor=cyan"
                     /note="/ApEinfo_graphicformat=arrow_data {{0 0.5 0 1 2 0 0 
                     -1 0
                     -0.5} {} 0} width 5 offset 0"
                     /note="/ApEinfo_label=Mutated BsmBI"
                     /note="/ApEinfo_revcolor=cyan"
     primer_bind     complement(5192..5218)
                     /label=TLP20_p12/13-PAC_R_72
     misc_feature    5215..5218
                     /label=p12/13-connector
     misc_signal     5221..5277
                     /locus_tag="P2A"
                     /label=P2A
     misc_feature    5280..5283
                     /locus_tag="p13-14 connector"
                     /label=p13-14 connector
     CDS             5284..5960
                     /locus_tag="tagBFP(1)"
                     /label=tagBFP
                     /label=tagBFP(1)
     CDS             5961..5982
                     /locus_tag="tagBFP"
                     /label=tagBFP
     misc_feature    5986..5989
                     /locus_tag="p14/15 connector"
                     /label=p14/15 connector
     polyA_site      6099..6150
                     /locus_tag="SPA"
                     /label=SPA
     misc_feature    6151..6271
                     /locus_tag="human complement component C2: MAZ 
                     terminatorbinding sequence"
                     /label=human complement component C2: MAZ
                     terminator b...
                     /label=human complement component C2: MAZ terminator
                     binding sequence
     primer_bind     complement(6195..6213)
                     /label=TLS15_C2MAZ_R_60
     misc_feature    6279..6282
                     /label=p15/16-connector
     misc_feature    6283..7496
                     /locus_tag="cHS4 Chicken B-globin insulator"
                     /label=cHS4 Chicken B-globin insulator
                     /note="cHS4 insulators are flanking the PUFFFIN cassette to
                     prevent gene silencing at the random integration site. This
                     will ensure stable PUFFFIN expression even when cells are 
                     cultured for a long time."
     misc_feature    7497..7500
                     /label=p25/V-connector
     terminator      complement(7574..7603)
                     /locus_tag="T3Te terminator"
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     primer_bind     complement(7589..7608)
                     /label=MMS150_EMMA_Receiver_2
     rep_origin      complement(7625..8212)
                     /direction=LEFT
                     /locus_tag="ori"
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7688..7707)
                     /label=MMS_EMMA_Reverse_New
     terminator      complement(8224..8251)
                     /locus_tag="T7Te terminator"
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     8224..8238
                     /label=MMS150_EMMA_Receiver_2
     CDS             complement(8275..9135)
                     /codon_start=1
                     /gene="bla"
                     /locus_tag="AmpR"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESLRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLATIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGSLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     misc_feature    8715..8720
                     /label=PvuI restriction site for linearisation
                     /note="PvuI restriction site for linearisation prior to 
                     transfection for random integration. This restriction site 
                     is far away from the PUFFFIN cassette coding region. 
                     Linearisation prior to transfection for making stable lines
                     is recommended to prevent double strang breaks in coding 
                     regions of the plasmid inside the cells. Linearisation 
                     should not be done for transient transfections."
     promoter        complement(9136..9238)
                     /locus_tag="cat promoter"
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     primer_bind     9223..9242
                     /label=MMS149_EMMA_Receiver_1
     terminator      complement(9239..9270)
                     /locus_tag="tonB terminator"
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription
                     terminator"
ORIGIN
        1 aatggtcagt attgagcgat atctagagaa ttcgtatagg gcgttaatta actagagccc
       61 catcctcact gactccgtcc tggagttgga tgagagataa tggccttacg ttgtgccagg
      121 ggagggtcgg gctggattta gcaagattta ccttctccaa agagcggtgc tgcagtggca
      181 cagctgccca cggaggtggg ggggtcaccg tccctggagg tgatgaagaa ctgtggggat
      241 gtggcactga gggacatggc cagtgggcac ggtgggtggg ttggggttgg tcttggggat
      301 cttggagggc ttttccagcc ttcatgattt gacgattgta tgaacatcta catggcaatt
      361 ctccagctgc ctgtcccagt cctactgacc cagctgtatc tctccaggca agctcttcca
      421 ccccttctgc ttgcatccag acaccatcaa acatgcaggc tcagacacag ggaccagcag
      481 tgtctgtggc ctttttgtgc tcctctccat gctgggtttt aacttgctct ttgtccttct
      541 atcctatctt cttatcctta aggctgttct gaacgctgtg acttggagag tgtcccagag
      601 ccctcaacac ctgcatgtcc cacgtccatg ctgtcctgca cttccttatc cccaagatct
      661 gcctctccgt gatgcactga attggcaaac atgtgtcacc ccagaccaac aatgtcacag
      721 caaactcccc cttgatagga caagggggaa tggctttaca ctgagacagg ggaggtttgg
      781 gttggatatg aggaggcagt ttttccccca gagggtggtg acgcactgaa caggttgccc
      841 aaggaggctg tggatgcccc atccctgcag gcattcaagg ccaggctgga tgtggctctg
      901 ggcagcctgg gctgctggtt gatgaccctg cacatagcag ggggttggat ctggatgagc
      961 actgtgctcc tttgcaaccc aggccgttct atgattctgt cattctaaat ctctctttca
     1021 gcctaaagct ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag
     1081 cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg
     1141 ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct
     1201 gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc
     1261 tctagggacg ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     1321 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     1381 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     1441 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     1501 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     1561 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     1621 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     1681 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     1741 gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg
     1801 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
     1861 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
     1921 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
     1981 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
     2041 tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt
     2101 gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt
     2161 gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg
     2221 cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt
     2281 gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg
     2341 ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct gcacccccct
     2401 ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg
     2461 gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg
     2521 cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt
     2581 cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga
     2641 cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct
     2701 agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     2761 cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg
     2821 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     2881 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     2941 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattcctcga ctccgcctag
     3001 gcgcgccacc tcgaggccgc caccatgaag tgggtgacct tcatcagcct gctgtttctg
     3061 ttcagcagcg cctactctag agggggcgga gcaagcaaag gcgagaggct gtttagaggg
     3121 aaagtgccaa tcctggtgga actgaaagga gacgtgaacg gccacaagtt ctctgtccga
     3181 ggcaagggga aaggagatgc cacacgggga aaactgactc tgaagtttat ctgcaccaca
     3241 ggcaagctgc ccgtgccttg gccaaccctg gtcactaccc tgacatacgg cgtgcagtgt
     3301 ttcagccgat atccaaaaca catgaagcgg catgacttct ttaaaagtgc tatgcccaag
     3361 ggctacgtgc aggagcgcac catttcattc aagaaggatg ggaagtacaa gacccgggcc
     3421 gaggtgaagt ttgaagggag aaccctggtc aaccgcatca agctgaaagg acgcgacttc
     3481 aaggaaaaag ggaatattct gggacacaaa ctgagataca acttcaacag ccataaggtg
     3541 tacatcacag cagataagag aaaaaacggc atcaaggcca aattcaagat taggcacaat
     3601 gtgaaggacg gctccgtcca gctggctgat cattaccagc agaacacacc tattggcaga
     3661 gggcccgtgc tgctgcctag gaatcattat ctgagcactc gctccaaact gtctaaggac
     3721 cccaaagaga agcgggatca catggtgctg ctggaatttg tcaccgccgc tggcatcaaa
     3781 catgggcggg acgagagata caagggcggc agcggcggca ccagcggcgg cggcggcggc
     3841 agcggcgtga gcaagggcga ggaggataac atggcctctc tcccagcgac acatgagtta
     3901 cacatctttg gctccatcaa cggtgtggac tttgacatgg tgggtcaggg caccggcaat
     3961 ccaaatgatg gttatgagga gttaaacctg aagtccacca agggtgacct ccagttctcc
     4021 ccctggattc tggtccctca tatcgggtat ggcttccatc agtacctgcc ctaccctgac
     4081 gggatgtcgc ctttccaggc cgccatggta gatggctccg gataccaagt ccatcgcaca
     4141 atgcagtttg aagatggtgc ctcccttact gttaactacc gctacaccta cgagggaagc
     4201 cacatcaaag gagaggccca ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg
     4261 accaactcgc tgaccgctgc ggactggtgc aggtcgaaga agacttaccc caacgacaaa
     4321 accatcatca gtacctttaa gtggagttac accactggaa atggcaagcg ctaccggagc
     4381 actgcgcgga ccacctacac ctttgccaag ccaatggcgg ctaactatct gaagaaccag
     4441 ccgatgtacg tgttccgtaa gacggagctc aagcactcca agaccgagct caacttcaag
     4501 gagtggcaaa aggcctttac cgatgtgatg ggcatggacg agctgtacaa gggtatagag
     4561 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc tggcgtcatg
     4621 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     4681 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     4741 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     4801 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     4861 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     4921 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     4981 gcgtggttcc tggccaccgt cggtgtgtcg cccgaccacc agggcaaggg tctgggcagc
     5041 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     5101 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     5161 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctcactt
     5221 gccacaaact tctctctgct aaagcaagca ggtgatgttg aagaaaaccc cgggcctgcc
     5281 tacatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
     5341 gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     5401 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct
     5461 actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc
     5521 ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg
     5581 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     5641 aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc
     5701 tgggaggcct tcaccgaaac gctgtacccc gctgacggcg gcctggaagg cagaaacgac
     5761 atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac cacatataga
     5821 tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg
     5881 gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc
     5941 agatactgcg acctccctag caaactgggg cacaagctta attgagcaaa tgacccgcaa
     6001 gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc
     6061 ccatgcatcg atgatctaga actagtggat ccccgatcca ataaaagatc tttattttca
     6121 ttagatctgt gtgttggttt tttgtgtggg cttgggggag ggggaggcca gaatgactcc
     6181 aagagctaca ggaaggcagg tcagagaccc cactggacaa acagtggctg gactctgcac
     6241 cataacacac aatcaacagg ggagtgagct ggatccggcc ctctagagcc ccatcctcac
     6301 tgactccgtc ctggagttgg atgagagata atggccttac gttgtgccag gggagggtcg
     6361 ggctggattt agcaagattt accttctcca aagagcggtg ctgcagtggc acagctgccc
     6421 acggaggtgg gggggtcacc gtccctggag gtgatgaaga actgtgggga tgtggcactg
     6481 agggacatgg ccagtgggca cggtgggtgg gttggggttg gtcttgggga tcttggaggg
     6541 cttttccagc cttcatgatt tgacgattgt atgaacatct acatggcaat tctccagctg
     6601 cctgtcccag tcctactgac ccagctgtat ctctccaggc aagctcttcc accccttctg
     6661 cttgcatcca gacaccatca aacatgcagg ctcagacaca gggaccagca gtgtctgtgg
     6721 cctttttgtg ctcctctcca tgctgggttt taacttgctc tttgtccttc tatcctatct
     6781 tcttatcctt aaggctgttc tgaacgctgt gacttggaga gtgtcccaga gccctcaaca
     6841 cctgcatgtc ccacgtccat gctgtcctgc acttccttat ccccaagatc tgcctctccg
     6901 tgatgcactg aattggcaaa catgtgtcac cccagaccaa caatgtcaca gcaaactccc
     6961 ccttgatagg acaaggggga atggctttac actgagacag gggaggtttg ggttggatat
     7021 gaggaggcag tttttccccc agagggtggt gacgcactga acaggttgcc caaggaggct
     7081 gtggatgccc catccctgca ggcattcaag gccaggctgg atgtggctct gggcagcctg
     7141 ggctgctggt tgatgaccct gcacatagca gggggttgga tctggatgag cactgtgctc
     7201 ctttgcaacc caggccgttc tatgattctg tcattctaaa tctctctttc agcctaaagc
     7261 tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag
     7321 gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg
     7381 ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag
     7441 cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc ctctagacga
     7501 taacgaattc aagcttgata tcattcagga cgagcctcag actccagcgt aactggactg
     7561 caatcaactc actggctcac cttcacgggt gggcctttct tcggtagaaa atcaaaggat
     7621 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7681 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aggtaactgg
     7741 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     7801 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7861 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7921 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7981 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     8041 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     8101 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     8161 acttgagcat cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     8221 caacgcagaa aggcccaccc gaaggtgagc caggtgatta catttgggcc ctcattacca
     8281 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     8341 ctgactcccc gtcgtgtaga taactacgat gcgggagggc ttaccatctg gccccagtgc
     8401 tgcaatgata ccgcgagaac cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     8461 agccgggagg gccgagcgca gaagtgatcc tgcaacttta tccgcctcca tccagtctat
     8521 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     8581 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     8641 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     8701 ctccttcggt cctccgatcg ttgccagaag taagttggcc gcagtgttat cactcatggt
     8761 tatggcagca ctgcataatt ctcttactgt catgccatcc gtgagatgct tttctgtgac
     8821 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     8881 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     8941 tggaaaacgt tcttcggggc gtaaactctc aaggatctta ccgctgttga gatccagttc
     9001 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     9061 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     9121 atgttgaata ctcattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     9181 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaag
     9241 tcaaaagcct ccggtcggag gcttttgact ttctgctatg gaggtcaggt atgattta
//
