LOCUS       pTL5-EMMA-PUFFFI        9913 bp DNA     circular SYN 28-JUN-2024
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 9913)
  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 9913)
  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..9913
                     /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..2984
                     /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..1912
                     /locus_tag="Chiken beta Actin promoter"
                     /label=Chiken beta Actin promoter
     promoter        1914..2984
                     /locus_tag="Rabbit beta globin splice acceptor"
                     /label=Rabbit beta globin splice acceptor
     promoter        1914..2984
                     /locus_tag="CAG promoter"
                     /label=CAG promoter
     primer_bind     2891..2912
                     /label=TLS1_CAG_F_58C
     primer_bind     2936..2946
                     /label=TLS3_36GFP_F_62C
     primer_bind     2957..3032
                     /label=TLP7_OEP_F_68
     primer_bind     2957..3026
                     /label=TLP5_OEP_F_64
     primer_bind     2991..3014
                     /label=TLP8_4/5-AscI_F_68
     misc_feature    2991..2994
                     /label=p4/5-connector
     primer_bind     2996..3014
                     /label=TLP3_XhoI_F_64C
     primer_bind     3005..3033
                     /label=TLP1_s36GFP_F_72C
     misc_feature    3015..3023
                     /label=kozak
     CDS             join(3024..3077,3078..3083,3084..3803,3804..3845,
                     3846..4553)
                     /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     3353..3369
                     /label=TLS3_36GFP_F_62C
     primer_bind     complement(3452..3472)
                     /label=TLS4_36GFP_R_61C
     primer_bind     3835..3861
                     /label=TLP12_p6/7-mCherry_F_73
     primer_bind     3846..3861
                     /label=TLP21_p13/14-mCherry_F_63
     primer_bind     3933..3952
                     /label=TLS7_mNG_F_63C
     primer_bind     complement(4024..4045)
                     /label=TLS8_mNG_R_55C
     primer_bind     complement(4538..4561)
                     /label=TLP9_p5/6-NotI_R_66
     misc_feature    4554..4561
                     /label=NotI restriction site
                     /note="NotI restriction site for future cloning without 
                     mCherry-3NLS into random integration plasmids MMP048 and 
                     MMP049."
     primer_bind     4557..4591
                     /label=TLP10_p5/6-IRES_F_63
     misc_feature    4562..5112
                     /gene="IRES"
                     /locus_tag="IRES"
                     /label=IRES
                     /note="IRES for strong separation of tandem constructs 
                     36GFP-HaloTag and mCherry-3NLS. This is to avoid false 
                     negatives: if using a p2A instead of IRES the highly 
                     positive charge of s36GFP-HT might interfere with 
                     self-cleavage, so mKate2 would also be secreted as part of 
                     a big fusion protein (s36GFP-HT-p2A-mCherry-3NLS), which 
                     could lead to labelled receivers being mistaken for 
                     secretors (= false negatives) or the system would not work 
                     at all because the big fusion protein cannot be secreted 
                     and taken up. An IRES to separate s36GFP-HT and 
                     mCherry-3NLS is the safest option."
     primer_bind     complement(4662..4683)
                     /label=TLS2_CAG_R_60C
     primer_bind     4927..4946
                     /label=TLS9_IRES_F_62C
     primer_bind     complement(5044..5064)
                     /label=TLS10_IRES_R_66C
     misc_feature    5113..5116
                     /label=p11/12-connector
     CDS             5117..5713
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="Confers resistance to antibiotic puromycin which 
                     allows comparably fast selection of clones with stable 
                     integration of the PUFFFIN cassette.
                     
                     The resistance gene in EMMA position 12 can be replaced 
                     with a transgene for GOI overexpression in combination with
                     PUFFFIN labelling. Correct stable clones will have to be 
                     isolated by fluorescence activated cell sorting. "
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5236..5256)
                     /label=TLS11_PAC_R_67C
     primer_bind     complement(5252..5271)
                     /label=MMS139_Pac_150R
     misc_feature    5503..5508
                     /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"
     misc_feature    5503..5506
                     /locus_tag="Mutated BsmBI connector"
                     /label=Mutated BsmBI connector
                     /note="/ApEinfo_fwdcolor=#ffff80"
                     /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 connector"
                     /note="/ApEinfo_revcolor=#ffff00"
     misc_feature    5714..5717
                     /locus_tag="p12-13 connector"
                     /label=p12-13 connector
     misc_signal     5720..5776
                     /locus_tag="P2A"
                     /label=P2A
                     /note="P2A self cleaving peptide for separation of the 
                     PuroR resistance gene and mCherry-3NLS.
                     
                     The P2A EMMA part has an extra 2 nucleotides before P2A 
                     sequence to keep the sequence in frame after the p12/13 4 
                     nucleotide connector (tcacTT --> SL). Has an extra 2 
                     nucleotides after P2A sequence to keep the sequence in 
                     frame before the p13/14 4 nucleotide connector (GCctac --> 
                     AY)."
     primer_bind     5777..5801
                     /label=TLP12_p6/7-mCherry_F_73
     primer_bind     5779..5801
                     /label=TLP21_p13/14-mCherry_F_63
     misc_feature    5779..5782
                     /label=p13/14-connector
     CDS             join(5783..6490,6491..6511,6512..6583)
                     /label=mCherry-3NLS
                     /note="mCherry as fluorescent marker that remains in the 
                     secretors to distinguish them from labelled acceptors. 
                     mCherry was chosen over mKate2 because pilot experiments 
                     showed that the latter was too bright and there was 
                     bleed-through into the green fluorescent channel.
                     
                     mCherry is downstream of the resistance gene in p12, that 
                     could be replaced with a transgene, to give a read-out for 
                     transgene expression (when mCherry is there, the transgene 
                     is being expressed, too, because it comes before mCherry). 
                     
                     3NLS are required to ensure that mCherry is entirely 
                     located to the nucleus and is not being transferred to 
                     other cells. Pilot experiments showed that mCherry-1NLS has
                     nuclear and cytoplasmic localisation and causes unspecific 
                     cross-labelling as seen by flow cytometry."
     misc_feature    5783..6490
                     /locus_tag="mCherry"
                     /label=mCherry
                     /note="mCherry as fluorescent marker that remains in the 
                     secretors to distinguish them from labelled acceptors. 
                     mCherry was chosen over mKate2 because pilot experiments 
                     showed that the latter was too bright and there was 
                     bleed-through into the green fluorescent channel.
                     
                     mCherry is downstream of the resistance gene in p12, that 
                     could be replaced with a transgene, to give a read-out for 
                     transgene expression (when mCherry is there, the transgene 
                     is being expressed, too, because it comes before mCherry). 
                     "
     primer_bind     complement(5962..5981)
                     /label=MMS123_mCherry_180R
     primer_bind     6369..6388
                     /label=MMS122_mCherry_600F
     primer_bind     complement(6406..6425)
                     /label=MMS124_mCherry_620R
     primer_bind     complement(6478..6490)
                     /label=TLP9_p5/6-NotI_R_66
     CDS             6491..6511
                     /locus_tag="Linker"
                     /label=Linker
     misc_signal     6512..6563
                     /locus_tag="3xNLS"
                     /label=3xNLS
                     /note="3NLS are required to ensure that mCherry is entirely
                     located to the nucleus and is not being transferred to 
                     other cells. Pilot experiments showed that mCherry-1NLS has
                     nuclear and cytoplasmic localisation and causes unspecific 
                     cross-labelling as seen by flow cytometry."
     misc_signal     6564..6583
                     /locus_tag="NLS"
                     /label=NLS
     primer_bind     complement(6581..6604)
                     /label=MMC425_CAG-backbone_EMMA-p14_R
     misc_feature    6601..6604
                     /locus_tag="p14-15 connector"
                     /label=p14-15 connector
     polyA_site      6714..6765
                     /locus_tag="SPA"
                     /label=SPA
     misc_feature    6766..6886
                     /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(6810..6828)
                     /label=TLS15_C2MAZ_R_60
     misc_feature    6894..6897
                     /label=p15/16-connector
     misc_feature    6898..8111
                     /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    8112..8115
                     /label=p25/V-connector
     terminator      complement(8189..8218)
                     /locus_tag="T3Te terminator"
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     primer_bind     complement(8204..8223)
                     /label=MMS150_EMMA_Receiver_2
     rep_origin      complement(8240..8827)
                     /direction=LEFT
                     /locus_tag="ori"
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8303..8322)
                     /label=MMS_EMMA_Reverse_New
     terminator      complement(8839..8866)
                     /locus_tag="T7Te terminator"
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     8839..8853
                     /label=MMS150_EMMA_Receiver_2
     CDS             complement(8890..9750)
                     /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    9330..9335
                     /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(9751..9853)
                     /locus_tag="cat promoter"
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     primer_bind     9838..9857
                     /label=MMS149_EMMA_Receiver_1
     terminator      complement(9854..9885)
                     /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 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
     1801 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     1861 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
     1921 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     1981 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     2041 gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg
     2101 aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg
     2161 tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc
     2221 gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg
     2281 ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg
     2341 gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc
     2401 cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg
     2461 ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc
     2521 ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc
     2581 gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac
     2641 ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta
     2701 gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc
     2761 gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg
     2821 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     2881 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     2941 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgac tccgcctagg
     3001 cgcgccacct cgaggccgcc accatgaagt gggtgacctt catcagcctg ctgtttctgt
     3061 tcagcagcgc ctactctaga gggggcggag caagcaaagg cgagaggctg tttagaggga
     3121 aagtgccaat cctggtggaa ctgaaaggag acgtgaacgg ccacaagttc tctgtccgag
     3181 gcaaggggaa aggagatgcc acacggggaa aactgactct gaagtttatc tgcaccacag
     3241 gcaagctgcc cgtgccttgg ccaaccctgg tcactaccct gacatacggc gtgcagtgtt
     3301 tcagccgata tccaaaacac atgaagcggc atgacttctt taaaagtgct atgcccaagg
     3361 gctacgtgca ggagcgcacc atttcattca agaaggatgg gaagtacaag acccgggccg
     3421 aggtgaagtt tgaagggaga accctggtca accgcatcaa gctgaaagga cgcgacttca
     3481 aggaaaaagg gaatattctg ggacacaaac tgagatacaa cttcaacagc cataaggtgt
     3541 acatcacagc agataagaga aaaaacggca tcaaggccaa attcaagatt aggcacaatg
     3601 tgaaggacgg ctccgtccag ctggctgatc attaccagca gaacacacct attggcagag
     3661 ggcccgtgct gctgcctagg aatcattatc tgagcactcg ctccaaactg tctaaggacc
     3721 ccaaagagaa gcgggatcac atggtgctgc tggaatttgt caccgccgct ggcatcaaac
     3781 atgggcggga cgagagatac aagggcggca gcggcggcac cagcggcggc ggcggcggca
     3841 gcggcgtgag caagggcgag gaggataaca tggcctctct cccagcgaca catgagttac
     3901 acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc accggcaatc
     3961 caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc cagttctccc
     4021 cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc taccctgacg
     4081 ggatgtcgcc tttccaggcc gccatggtag atggctccgg ataccaagtc catcgcacaa
     4141 tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac gagggaagcc
     4201 acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt cctgtgatga
     4261 ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc aacgacaaaa
     4321 ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc taccggagca
     4381 ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg aagaaccagc
     4441 cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc aacttcaagg
     4501 agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag taagcggccg
     4561 cttgatatcg aattccgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc
     4621 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     4681 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     4741 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     4801 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     4861 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     4921 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     4981 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     5041 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaacgtctag
     5101 gccccccgaa cccgtcatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     5161 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     5221 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     5281 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     5341 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     5401 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     5461 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggtgtgtcgc ccgaccacca
     5521 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     5581 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     5641 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     5701 caagcccggt gcctcacttg ccacaaactt ctctctgcta aagcaagcag gtgatgttga
     5761 agaaaacccc gggcctgcct acatggtgag caagggcgag gaggataaca tggccatcat
     5821 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     5881 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     5941 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     6001 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     6061 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     6121 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     6181 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
     6241 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     6301 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     6361 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     6421 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     6481 gctgtacaag acctcaggca ggacaggtag tccaaaaaag aagagaaagg tagatccaaa
     6541 aaagaagaga aaggtagatc caaaaaagaa gagaaaggta ttataagcgg ccgctcgata
     6601 gcaaatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga
     6661 aaggagcgca cgaccccatg catcgatgat ctagaactag tggatccccg atccaataaa
     6721 agatctttat tttcattaga tctgtgtgtt ggttttttgt gtgggcttgg gggaggggga
     6781 ggccagaatg actccaagag ctacaggaag gcaggtcaga gaccccactg gacaaacagt
     6841 ggctggactc tgcaccataa cacacaatca acaggggagt gagctggatc cggccctcta
     6901 gagccccatc ctcactgact ccgtcctgga gttggatgag agataatggc cttacgttgt
     6961 gccaggggag ggtcgggctg gatttagcaa gatttacctt ctccaaagag cggtgctgca
     7021 gtggcacagc tgcccacgga ggtggggggg tcaccgtccc tggaggtgat gaagaactgt
     7081 ggggatgtgg cactgaggga catggccagt gggcacggtg ggtgggttgg ggttggtctt
     7141 ggggatcttg gagggctttt ccagccttca tgatttgacg attgtatgaa catctacatg
     7201 gcaattctcc agctgcctgt cccagtccta ctgacccagc tgtatctctc caggcaagct
     7261 cttccacccc ttctgcttgc atccagacac catcaaacat gcaggctcag acacagggac
     7321 cagcagtgtc tgtggccttt ttgtgctcct ctccatgctg ggttttaact tgctctttgt
     7381 ccttctatcc tatcttctta tccttaaggc tgttctgaac gctgtgactt ggagagtgtc
     7441 ccagagccct caacacctgc atgtcccacg tccatgctgt cctgcacttc cttatcccca
     7501 agatctgcct ctccgtgatg cactgaattg gcaaacatgt gtcaccccag accaacaatg
     7561 tcacagcaaa ctcccccttg ataggacaag ggggaatggc tttacactga gacaggggag
     7621 gtttgggttg gatatgagga ggcagttttt cccccagagg gtggtgacgc actgaacagg
     7681 ttgcccaagg aggctgtgga tgccccatcc ctgcaggcat tcaaggccag gctggatgtg
     7741 gctctgggca gcctgggctg ctggttgatg accctgcaca tagcaggggg ttggatctgg
     7801 atgagcactg tgctcctttg caacccaggc cgttctatga ttctgtcatt ctaaatctct
     7861 ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc
     7921 gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc
     7981 acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc
     8041 gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc
     8101 tgtccctcta gacgataacg aattcaagct tgatatcatt caggacgagc ctcagactcc
     8161 agcgtaactg gactgcaatc aactcactgg ctcaccttca cgggtgggcc tttcttcggt
     8221 agaaaatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     8281 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     8341 ttccgaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     8401 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     8461 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     8521 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     8581 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     8641 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     8701 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     8761 gtttcgccac ctctgacttg agcatcgatt tttgtgatgc tcgtcagggg ggcggagcct
     8821 atggaaaaac gccagcaacg cagaaaggcc cacccgaagg tgagccaggt gattacattt
     8881 gggccctcat taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     8941 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatgcggg agggcttacc
     9001 atctggcccc agtgctgcaa tgataccgcg agaaccacgc tcaccggctc cagatttatc
     9061 agcaataaac cagccagccg ggagggccga gcgcagaagt gatcctgcaa ctttatccgc
     9121 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     9181 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     9241 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     9301 caaaaaagcg gttagctcct tcggtcctcc gatcgttgcc agaagtaagt tggccgcagt
     9361 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtgag
     9421 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     9481 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     9541 aaaagtgctc atcattggaa aacgttcttc ggggcgtaaa ctctcaagga tcttaccgct
     9601 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     9661 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     9721 aagggcgaca cggaaatgtt gaatactcat tttagcttcc ttagctcctg aaaatctcga
     9781 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     9841 tacgtgccga tcaagtcaaa agcctccggt cggaggcttt tgactttctg ctatggaggt
     9901 caggtatgat tta
//
