LOCUS       pTL52-EMMA-PUFFL        7585 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 7585)
  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 7585)
  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..7585
                     /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     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
     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..4011)
                     /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="HaloTag (Los et al. ACS Chemical Biology 3 (6): 
                     373–82 (2008)) with stop codon. HaloTag was chosen as a 
                     label amplifier for colour of choice labelling. +36GFP will
                     be able to drag HaloTag through the cell membrane to enable
                     uptake as shown for mCherry by Cronican et al. ACS Chem. 
                     Biol. 5, 747–752 (2010)."
                     /label=s36GFP-HaloTag tandem construct
                     /translation="MKWVTFISLLFLFSSAYSRGGGASKGERLFRGKVPILVELKGDVN
                     GHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHD
                     FFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKL
                     RYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHY
                     LSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKGGSGGTSGGGGGSGAEIGTGF
                     PFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLI
                     GMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGI
                     AFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVE
                     MDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPG
                     VLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG"
     primer_bind     2628..2644
                     /label=TLS3_36GFP_F_62C
     primer_bind     2628..2644
                     /label=TLS3_36GFP_F_62Ca
     primer_bind     complement(2727..2747)
                     /label=TLS4_36GFP_R_61C
     primer_bind     3094..3138
                     /label=OEP_HaloTag_F
     primer_bind     complement(3094..3137)
                     /label=OEP_14_long
     CDS             3121..4008
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     primer_bind     3975..3984
                     /label=TLS17_Myod1_R_67
     primer_bind     complement(3989..4023)
                     /label=Halotag-NotI
     primer_bind     4012..4030
                     /label=TLP9_p5/6-NotI_R_66
     primer_bind     4012..4030
                     /label=TLP9_p5/6-NotI_R_66a
     primer_bind     4012..4030
                     /label=TLP9_p5/6-NotI_R
     misc_feature    4020..4023
                     /label=p10/11 connector
     CDS             4133..4632
                     /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     4407..4426
                     /label=TLS9_IRES_F_62C
     primer_bind     complement(4524..4544)
                     /label=TLS10_IRES_R_66C
     primer_bind     complement(4575..4593)
                     /label=TLP11_p6/7-IRES_R_63
     primer_bind     complement(4610..4637)
                     /label=MMC419_IRES_EMMA-p11_R
     primer_bind     4633..4664
                     /label=TLP37_p11/12-mCherry_F_73
     misc_feature    4633..4636
                     /label=p11/12-connector
     primer_bind     4636..4664
                     /label=TLP12_p6/7-mCherry_F_73
     primer_bind     4636..4664
                     /label=p6/7-mCherry
     primer_bind     4637..4648
                     /label=TLP5_OEP_F_64
     misc_feature    4637..4645
                     /label=kozak
     primer_bind     4645..4664
                     /label=TLP21_p13/14-mCherry_F_63
     primer_bind     4645..4664
                     /label=TLP21_p13/14-mCherry_F_63a
     primer_bind     4645..4664
                     /label=TLP21_p13/14-mCherry_F_63`b
     primer_bind     4645..4664
                     /label=TLP21_p13/14-mCherry_F_63`c
     CDS             join(4646..5353,5354..5374,5375..5449)
                     /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    4646..5353
                     /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(4825..4844)
                     /label=MMS123_mCherry_180R
     primer_bind     5232..5251
                     /label=MMS122_mCherry_600F
     primer_bind     complement(5269..5288)
                     /label=MMS124_mCherry_620R
     primer_bind     complement(5341..5353)
                     /label=TLP9_p5/6-NotI_R_66
     primer_bind     complement(5341..5353)
                     /label=TLP9_p5/6-NotI_R_66a
     primer_bind     complement(5341..5353)
                     /label=TLP9_p5/6-NotI_R
     CDS             5354..5374
                     /locus_tag="Linker"
                     /label=Linker
     misc_signal     5375..5426
                     /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(5416..5461)
                     /label=p7/8-NLS
     misc_signal     5427..5446
                     /locus_tag="NLS"
                     /label=NLS
     primer_bind     complement(5444..5467)
                     /label=MMC425_CAG-backbone_EMMA-p14_R
     misc_feature    5464..5467
                     /label=p14/15-connector
     polyA_site      5577..5628
                     /locus_tag="SPA"
                     /label=SPA
     misc_feature    5629..5749
                     /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(5673..5691)
                     /label=TLS15_C2MAZ_R_60
     misc_feature    5757..5760
                     /label=p15/16-connector
     misc_feature    5784..5787
                     /label=p25/V-connector
     terminator      complement(5861..5890)
                     /locus_tag="T3Te terminator"
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     primer_bind     complement(5876..5895)
                     /label=MMS150_EMMA_Receiver_2
     rep_origin      complement(5912..6499)
                     /direction=LEFT
                     /locus_tag="ori"
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5975..5994)
                     /label=MMS_EMMA_Reverse_New
     terminator      complement(6511..6538)
                     /locus_tag="T7Te terminator"
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     6511..6525
                     /label=MMS150_EMMA_Receiver_2
     CDS             complement(6562..7422)
                     /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    7002..7007
                     /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(7423..7525)
                     /locus_tag="cat promoter"
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     primer_bind     7510..7529
                     /label=MMS149_EMMA_Receiver_1
     terminator      complement(7526..7557)
                     /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 gcagaaatcg gtactggctt tccattcgac ccccattatg tggaagtcct gggcgagcgc
     3181 atgcactacg tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct gcacggtaac
     3241 ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc
     3301 attgctccag acctgatcgg tatgggcaaa tccgacaaac cagacctggg ttatttcttc
     3361 gacgaccacg tccgcttcat ggatgccttc atcgaagccc tgggtctgga agaggtcgtc
     3421 ctggtcattc acgactgggg ctccgctctg ggtttccact gggccaagcg caatccagag
     3481 cgcgtcaaag gtattgcatt tatggagttc atccgcccta tcccgacctg ggacgaatgg
     3541 ccagaatttg cccgcgagac cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg
     3601 atcatcgatc agaacgtttt tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg
     3661 actgaagtcg agatggacca ttaccgcgag ccgttcctga atcctgttga ccgcgagcca
     3721 ctgtggcgct tcccaaacga gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg
     3781 gtcgaagaat acatggactg gctgcaccag tcccctgtcc cgaagctgct gttctggggc
     3841 accccaggcg ttctgatccc accggccgaa gccgctcgcc tggccaaaag cctgcctaac
     3901 tgcaaggctg tggacatcgg cccgggtctg aatctgctgc aagaagacaa cccggacctg
     3961 atcggcagcg agatcgcgcg ctggctgtcg acgctggaga tttccggcta agcggccgcg
     4021 gtaccgctcg ataagcttga tatcgaattc cgcccccccc cctctccctc ccccccccta
     4081 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     4141 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     4201 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     4261 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     4321 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     4381 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     4441 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     4501 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     4561 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     4621 cacgatgata atcgtcgccg ccaccatggt gagcaagggc gaggaggata acatggccat
     4681 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     4741 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     4801 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     4861 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     4921 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     4981 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     5041 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc
     5101 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     5161 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     5221 gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga
     5281 ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga
     5341 cgagctgtac aagacctcag gcaggacagg tagtccaaaa aagaagagaa aggtagatcc
     5401 aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag gtattataag cggccgctcg
     5461 atagcaaatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac
     5521 cgaaaggagc gcacgacccc atgcatcgat gatctagaac tagtggatcc ccgatccaat
     5581 aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgggct tgggggaggg
     5641 ggaggccaga atgactccaa gagctacagg aaggcaggtc agagacccca ctggacaaac
     5701 agtggctgga ctctgcacca taacacacaa tcaacagggg agtgagctgg atccggccct
     5761 ataaggcgcg ccgtgcgata ataacgataa cgaattcaag cttgatatca ttcaggacga
     5821 gcctcagact ccagcgtaac tggactgcaa tcaactcact ggctcacctt cacgggtggg
     5881 cctttcttcg gtagaaaatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5941 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6001 taccaactct ttttccgagg taactggctt cagcagagcg cagataccaa atactgttct
     6061 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6121 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6181 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6241 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6301 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6361 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6421 tagtcctgtc gggtttcgcc acctctgact tgagcatcga tttttgtgat gctcgtcagg
     6481 ggggcggagc ctatggaaaa acgccagcaa cgcagaaagg cccacccgaa ggtgagccag
     6541 gtgattacat ttgggccctc attaccaatg cttaatcagt gaggcaccta tctcagcgat
     6601 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatgcg
     6661 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagaaccac gctcaccggc
     6721 tccagattta tcagcaataa accagccagc cgggagggcc gagcgcagaa gtgatcctgc
     6781 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6841 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6901 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6961 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg ccagaagtaa
     7021 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7081 gccatccgtg agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7141 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7201 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgta aactctcaag
     7261 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7321 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7381 aaaaaaggga ataagggcga cacggaaatg ttgaatactc attttagctt ccttagctcc
     7441 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa
     7501 gttggaacct cttacgtgcc gatcaagtca aaagcctccg gtcggaggct tttgactttc
     7561 tgctatggag gtcaggtatg attta
//
