LOCUS       pTL53-EMMA-PUFFL        7340 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 7340)
  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 7340)
  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..7340
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    37..40
                     /label=pV/1-connector
     misc_feature    73..76
                     /locus_tag="p2-3 connector"
                     /label=p2-3 connector
     enhancer        78..271
                     /locus_tag="PyF101"
                     /label=PyF101
     misc_feature    complement(272..359)
                     /locus_tag="Py ori"
                     /label=Py ori
     polyA_signal    392..446
                     /locus_tag="SV40pA"
                     /label=SV40pA
     polyA_site      442..488
                     /locus_tag="SV40pA core"
                     /label=SV40pA core
     misc_feature    540..543
                     /label=p3/4-connector
     promoter        605..2259
                     /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        605..910
                     /locus_tag="CMV enhancer"
                     /label=CMV enhancer
     primer_bind     complement(616..635)
                     /label=MMS140_CAG-50R
     primer_bind     complement(812..828)
                     /label=MMS140_CAG-50R
     promoter        911..1187
                     /locus_tag="Chiken beta Actin promoter"
                     /label=Chiken beta Actin promoter
     promoter        1189..2259
                     /locus_tag="Rabbit beta globin splice acceptor"
                     /label=Rabbit beta globin splice acceptor
     promoter        1189..2259
                     /locus_tag="CAG promoter"
                     /label=CAG promoter
     primer_bind     2037..2078
                     /label=Halotag-NotI
     primer_bind     2166..2187
                     /label=TLS1_CAG_F_58C
     primer_bind     2211..2221
                     /label=TLS3_36GFP_F_62C
     primer_bind     2211..2221
                     /label=TLS3_36GFP_F_62Ca
     primer_bind     2211..2221
                     /label=TLS3_36GFP_F_62C`b
     primer_bind     2232..2307
                     /label=TLP7_OEP_F_68
     primer_bind     2232..2301
                     /label=TLP5_OEP_F_64
     primer_bind     2266..2289
                     /label=TLP8_4/5-AscI_F_68
     primer_bind     2266..2289
                     /label=TLP8_4/5-AscI_F_68a
     primer_bind     2266..2289
                     /label=TLP8_4/5-AscI_F_68`b
     misc_feature    2266..2269
                     /label=p4/5-connector
     primer_bind     2269..2289
                     /label=TLP36_1/2-AscI_F_68
     primer_bind     2269..2289
                     /label=TLP41_5/6-AscI_F_68
     primer_bind     2271..2289
                     /label=TLP3_XhoI_F_64C
     primer_bind     2280..2308
                     /label=TLP1_s36GFP_F_72C
     misc_feature    2290..2298
                     /label=kozak
     CDS             join(2299..2352,2353..2358,2359..3078,3079..3120,
                     3121..3828)
                     /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     2628..2644
                     /label=TLS3_36GFP_F_62C
     primer_bind     2628..2644
                     /label=TLS3_36GFP_F_62Ca
     primer_bind     2628..2644
                     /label=TLS3_36GFP_F_62C`b
     primer_bind     complement(2727..2747)
                     /label=TLS4_36GFP_R_61C
     primer_bind     complement(3094..3121)
                     /label=OEP_14_long
     primer_bind     3094..3121
                     /label=OEP_HaloTag_F
     primer_bind     complement(3094..3121)
                     /label=TLP48_OEP_14-HT_R_83
     primer_bind     3110..3136
                     /label=TLP12_p6/7-mCherry_F_73
     primer_bind     3110..3136
                     /label=p6/7-mCherry
     primer_bind     complement(3113..3127)
                     /label=OEP_14_R
     primer_bind     3121..3136
                     /label=TLP21_p13/14-mCherry_F_63
     primer_bind     3121..3136
                     /label=TLP21_p13/14-mCherry_F_63a
     primer_bind     3121..3136
                     /label=TLP21_p13/14-mCherry_F_63`b
     primer_bind     3121..3136
                     /label=TLP37_p11/12-mCherry_F_73
     primer_bind     3121..3136
                     /label=TLP21_p13/14-mCherry_F_63`c
     primer_bind     3208..3227
                     /label=TLS7_mNG_F_63C
     primer_bind     complement(3299..3320)
                     /label=TLS8_mNG_R_55C
     primer_bind     complement(3813..3836)
                     /label=TLP9_p5/6-NotI_R_66
     primer_bind     complement(3813..3836)
                     /label=TLP9_p5/6-NotI_R_66a
     primer_bind     complement(3813..3836)
                     /label=TLP9_p5/6-NotI_R
     misc_feature    3829..3836
                     /label=NotI restriction site
                     /note="NotI restriction site for future cloning without 
                     mKate2 (if needed) into random integration plasmids MMP048 
                     and MMP049."
     primer_bind     3832..3866
                     /label=TLP10_p5/6-IRES_F_63
     misc_feature    3837..4387
                     /gene="IRES"
                     /locus_tag="IRES"
                     /label=IRES
                     /note="IRES for strong separation of tandem constructs 
                     36GFP-mNG and mKate2-3NLS. This is to avoid false 
                     negatives: if using a p2A instead of IRES the highly 
                     positive charge of s36GFP-mNG might interfere with 
                     self-cleavage, so mKate2 would also be secreted as part of 
                     a big fusion protein (s36GFP-mNG-p2A-mKate2), 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-mNG and mKate2-3NLS is the 
                     safest option."
     primer_bind     complement(3937..3958)
                     /label=TLS2_CAG_R_60C
     primer_bind     4202..4221
                     /label=TLS9_IRES_F_62C
     primer_bind     complement(4319..4339)
                     /label=TLS10_IRES_R_66C
     primer_bind     complement(4369..4392)
                     /label=p11/12-IRES
     primer_bind     complement(4369..4388)
                     /label=TLP11_p6/7-IRES_R_63
     primer_bind     complement(4369..4388)
                     /label=p6/7-IRES
     primer_bind     4388..4419
                     /label=TLP37_p11/12-mCherry_F_73
     misc_feature    4388..4391
                     /label=p11/12-connector
     primer_bind     4391..4419
                     /label=TLP12_p6/7-mCherry_F_73
     primer_bind     4391..4419
                     /label=p6/7-mCherry
     primer_bind     4392..4403
                     /label=TLP5_OEP_F_64
     misc_feature    4392..4400
                     /label=kozak
     primer_bind     4400..4419
                     /label=TLP21_p13/14-mCherry_F_63
     primer_bind     4400..4419
                     /label=TLP21_p13/14-mCherry_F_63a
     primer_bind     4400..4419
                     /label=TLP21_p13/14-mCherry_F_63`b
     primer_bind     4400..4419
                     /label=TLP21_p13/14-mCherry_F_63`c
     CDS             join(4401..5108,5109..5129,5130..5204)
                     /codon_start=1
                     /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). 
                     "
                     /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."
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYKTSGRTGSPKKKRKVDPKKKRKVDPKKKRKVL"
     misc_feature    4401..5108
                     /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(4580..4599)
                     /label=MMS123_mCherry_180R
     primer_bind     4987..5006
                     /label=MMS122_mCherry_600F
     primer_bind     complement(5024..5043)
                     /label=MMS124_mCherry_620R
     primer_bind     complement(5096..5108)
                     /label=TLP9_p5/6-NotI_R_66
     primer_bind     complement(5096..5108)
                     /label=TLP9_p5/6-NotI_R_66a
     primer_bind     complement(5096..5108)
                     /label=TLP9_p5/6-NotI_R
     CDS             5109..5129
                     /locus_tag="Linker"
                     /label=Linker
     misc_signal     5130..5181
                     /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."
     primer_bind     complement(5171..5216)
                     /label=p7/8-NLS
     misc_signal     5182..5201
                     /locus_tag="NLS"
                     /label=NLS
     primer_bind     complement(5199..5222)
                     /label=MMC425_CAG-backbone_EMMA-p14_R
     misc_feature    5219..5222
                     /label=p14/15-connector
     polyA_site      5332..5383
                     /locus_tag="SPA"
                     /label=SPA
     misc_feature    5384..5504
                     /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(5428..5446)
                     /label=TLS15_C2MAZ_R_60
     misc_feature    5512..5515
                     /label=p15/16-connector
     misc_feature    5539..5542
                     /label=p25/V-connector
     terminator      complement(5616..5645)
                     /locus_tag="T3Te terminator"
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     primer_bind     complement(5631..5650)
                     /label=MMS150_EMMA_Receiver_2
     rep_origin      complement(5667..6254)
                     /direction=LEFT
                     /locus_tag="ori"
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5730..5749)
                     /label=MMS_EMMA_Reverse_New
     terminator      complement(6266..6293)
                     /locus_tag="T7Te terminator"
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     6266..6280
                     /label=MMS150_EMMA_Receiver_2
     CDS             complement(6317..7177)
                     /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    6757..6762
                     /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(7178..7280)
                     /locus_tag="cat promoter"
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     primer_bind     7265..7284
                     /label=MMS149_EMMA_Receiver_1
     terminator      complement(7281..7312)
                     /locus_tag="tonB terminator"
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription
                     terminator"
ORIGIN
        1 aatggtcagt attgagcgat atctagagaa ttcgtatagg agatctttaa ttaaacgcgt
       61 gctagcgtcg acgactcagc tgttgtcata gtaatgattg ggtggaaaca ttccaggcct
      121 gggtggagag gctttttgct tcctcttgca aaaccacact gacattccag gcctgggtgg
      181 agaggctttt tgcttcctct tgcaaaacca cactgccctc tggagggcag ttgcctagca
      241 actaattaaa agaggatgtc gcacggccag ctgcggtcag ttagtcactt cctgcttaac
      301 tgacttgaca ttttctattt taagagtcgg gaggaaaatt actgtgttgg aggccctccg
      361 ccatcttctg aagctgaatc gaattaactt gtttattgca gcttataatg gttacaaata
      421 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
      481 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgatatcatc gtcgacgacg
      541 gacgctagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      601 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      661 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      721 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      781 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      841 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      901 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
      961 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     1021 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
     1081 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
     1141 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
     1201 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     1261 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1321 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg
     1381 ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc
     1441 gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc
     1501 gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg
     1561 cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg
     1621 tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga
     1681 gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc
     1741 gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg
     1801 gccggggagg gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc
     1861 gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct
     1921 ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg
     1981 cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg
     2041 tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc
     2101 tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct
     2161 agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg
     2221 ctggttattg tgctgtctca tcattttggc aaagaattcc tcgactccgc ctaggcgcgc
     2281 cacctcgagg ccgccaccat gaagtgggtg accttcatca gcctgctgtt tctgttcagc
     2341 agcgcctact ctagaggggg cggagcaagc aaaggcgaga ggctgtttag agggaaagtg
     2401 ccaatcctgg tggaactgaa aggagacgtg aacggccaca agttctctgt ccgaggcaag
     2461 gggaaaggag atgccacacg gggaaaactg actctgaagt ttatctgcac cacaggcaag
     2521 ctgcccgtgc cttggccaac cctggtcact accctgacat acggcgtgca gtgtttcagc
     2581 cgatatccaa aacacatgaa gcggcatgac ttctttaaaa gtgctatgcc caagggctac
     2641 gtgcaggagc gcaccatttc attcaagaag gatgggaagt acaagacccg ggccgaggtg
     2701 aagtttgaag ggagaaccct ggtcaaccgc atcaagctga aaggacgcga cttcaaggaa
     2761 aaagggaata ttctgggaca caaactgaga tacaacttca acagccataa ggtgtacatc
     2821 acagcagata agagaaaaaa cggcatcaag gccaaattca agattaggca caatgtgaag
     2881 gacggctccg tccagctggc tgatcattac cagcagaaca cacctattgg cagagggccc
     2941 gtgctgctgc ctaggaatca ttatctgagc actcgctcca aactgtctaa ggaccccaaa
     3001 gagaagcggg atcacatggt gctgctggaa tttgtcaccg ccgctggcat caaacatggg
     3061 cgggacgaga gatacaaggg cggcagcggc ggcaccagcg gcggcggcgg cggcagcggc
     3121 gtgagcaagg gcgaggagga taacatggcc tctctcccag cgacacatga gttacacatc
     3181 tttggctcca tcaacggtgt ggactttgac atggtgggtc agggcaccgg caatccaaat
     3241 gatggttatg aggagttaaa cctgaagtcc accaagggtg acctccagtt ctccccctgg
     3301 attctggtcc ctcatatcgg gtatggcttc catcagtacc tgccctaccc tgacgggatg
     3361 tcgcctttcc aggccgccat ggtagatggc tccggatacc aagtccatcg cacaatgcag
     3421 tttgaagatg gtgcctccct tactgttaac taccgctaca cctacgaggg aagccacatc
     3481 aaaggagagg cccaggtgaa ggggactggt ttccctgctg acggtcctgt gatgaccaac
     3541 tcgctgaccg ctgcggactg gtgcaggtcg aagaagactt accccaacga caaaaccatc
     3601 atcagtacct ttaagtggag ttacaccact ggaaatggca agcgctaccg gagcactgcg
     3661 cggaccacct acacctttgc caagccaatg gcggctaact atctgaagaa ccagccgatg
     3721 tacgtgttcc gtaagacgga gctcaagcac tccaagaccg agctcaactt caaggagtgg
     3781 caaaaggcct ttaccgatgt gatgggcatg gacgagctgt acaagtaagc ggccgcttga
     3841 tatcgaattc cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga
     3901 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     3961 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     4021 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     4081 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     4141 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     4201 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     4261 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     4321 ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc
     4381 ccgaacccgt cgccgccacc atggtgagca agggcgagga ggataacatg gccatcatca
     4441 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     4501 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     4561 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     4621 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     4681 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     4741 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     4801 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
     4861 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     4921 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     4981 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     5041 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     5101 tgtacaagac ctcaggcagg acaggtagtc caaaaaagaa gagaaaggta gatccaaaaa
     5161 agaagagaaa ggtagatcca aaaaagaaga gaaaggtatt ataagcggcc gctcgatagc
     5221 aaatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa
     5281 ggagcgcacg accccatgca tcgatgatct agaactagtg gatccccgat ccaataaaag
     5341 atctttattt tcattagatc tgtgtgttgg ttttttgtgt gggcttgggg gagggggagg
     5401 ccagaatgac tccaagagct acaggaaggc aggtcagaga ccccactgga caaacagtgg
     5461 ctggactctg caccataaca cacaatcaac aggggagtga gctggatccg gccctataag
     5521 gcgcgccgtg cgataataac gataacgaat tcaagcttga tatcattcag gacgagcctc
     5581 agactccagc gtaactggac tgcaatcaac tcactggctc accttcacgg gtgggccttt
     5641 cttcggtaga aaatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5701 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5761 actctttttc cgaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5821 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5881 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5941 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6001 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6061 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6121 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6181 ctgtcgggtt tcgccacctc tgacttgagc atcgattttt gtgatgctcg tcaggggggc
     6241 ggagcctatg gaaaaacgcc agcaacgcag aaaggcccac ccgaaggtga gccaggtgat
     6301 tacatttggg ccctcattac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6361 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atgcgggagg
     6421 gcttaccatc tggccccagt gctgcaatga taccgcgaga accacgctca ccggctccag
     6481 atttatcagc aataaaccag ccagccggga gggccgagcg cagaagtgat cctgcaactt
     6541 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6601 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6661 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6721 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgccaga agtaagttgg
     6781 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6841 ccgtgagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6901 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6961 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgtaaactc tcaaggatct
     7021 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7081 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7141 agggaataag ggcgacacgg aaatgttgaa tactcatttt agcttcctta gctcctgaaa
     7201 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
     7261 aacctcttac gtgccgatca agtcaaaagc ctccggtcgg aggcttttga ctttctgcta
     7321 tggaggtcag gtatgattta
//
