LOCUS       pTL51-EMMA-PUFFH       10159 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 10159)
  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 10159)
  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..10159
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    37..40
                     /label=pV/1-connector
     misc_feature    52..1265
                     /locus_tag="cHS4 Chicken B-globin insulator"
                     /label=cHS4 Chicken B-globin insulator
                     /note="cHS4 insulators are flanking the PUFFFIN cassette to
                     prevent gene silencing at the random integration site. This
                     will ensure stable PUFFFIN expression even when cells are 
                     cultured for a long time."
     misc_feature    1266..1269
                     /label=p3/4-connector
     promoter        1331..2985
                     /locus_tag="CAG"
                     /label=CAG
                     /note="The CAG promoter is the strongest, ubiquitous 
                     promoter we have in the lab and should lead to high 
                     expression levels of the PUFFFIN cassette."
     enhancer        1331..1636
                     /locus_tag="CMV enhancer"
                     /label=CMV enhancer
     primer_bind     complement(1342..1361)
                     /label=MMS140_CAG-50R
     primer_bind     complement(1538..1554)
                     /label=MMS140_CAG-50R
     promoter        1637..1913
                     /locus_tag="Chiken beta Actin promoter"
                     /label=Chiken beta Actin promoter
     promoter        1915..2985
                     /locus_tag="Rabbit beta globin splice acceptor"
                     /label=Rabbit beta globin splice acceptor
     promoter        1915..2985
                     /locus_tag="CAG promoter"
                     /label=CAG promoter
     primer_bind     2763..2804
                     /label=Halotag-NotI
     primer_bind     2892..2913
                     /label=TLS1_CAG_F_58C
     primer_bind     2937..2947
                     /label=TLS3_36GFP_F_62C
     primer_bind     2937..2947
                     /label=TLS3_36GFP_F_62Ca
     primer_bind     2958..3033
                     /label=TLP7_OEP_F_68
     primer_bind     2958..3027
                     /label=TLP5_OEP_F_64
     primer_bind     2992..3015
                     /label=TLP8_4/5-AscI_F_68
     primer_bind     2992..3015
                     /label=TLP8_4/5-AscI_F_68a
     misc_feature    2992..2995
                     /label=p4/5-connector
     primer_bind     2995..3015
                     /label=TLP36_1/2-AscI_F_68
     primer_bind     2995..3015
                     /label=TLP41_5/6-AscI_F_68
     primer_bind     2997..3015
                     /label=TLP3_XhoI_F_64C
     primer_bind     3006..3034
                     /label=TLP1_s36GFP_F_72C
     misc_feature    3016..3024
                     /label=kozak
     CDS             join(3025..3078,3079..3084,3085..3804,3805..3846,
                     3847..4737)
                     /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     3354..3370
                     /label=TLS3_36GFP_F_62C
     primer_bind     3354..3370
                     /label=TLS3_36GFP_F_62Ca
     primer_bind     complement(3453..3473)
                     /label=TLS4_36GFP_R_61C
     primer_bind     3820..3864
                     /label=OEP_HaloTag_F
     primer_bind     complement(3820..3863)
                     /label=OEP_14_long
     CDS             3847..4734
                     /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     4701..4710
                     /label=TLS17_Myod1_R_67
     primer_bind     complement(4715..4749)
                     /label=Halotag-NotI
     primer_bind     4738..4756
                     /label=TLP9_p5/6-NotI_R_66
     misc_feature    4746..4749
                     /label=p10/11 connector
     CDS             4859..5358
                     /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     5133..5152
                     /label=TLS9_IRES_F_62C
     primer_bind     complement(5250..5270)
                     /label=TLS10_IRES_R_66C
     primer_bind     complement(5301..5319)
                     /label=TLP11_p6/7-IRES_R_63
     primer_bind     complement(5336..5362)
                     /label=MMC419_IRES_EMMA-p11_R
     misc_feature    5359..5362
                     /label=p11/12-connector
     CDS             5363..5959
                     /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(5482..5502)
                     /label=TLS11_PAC_R_67C
     primer_bind     complement(5498..5517)
                     /label=MMS139_Pac_150R
     misc_feature    5749..5754
                     /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    5749..5752
                     /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    5960..5963
                     /locus_tag="p12-13 connector"
                     /label=p12-13 connector
     misc_signal     5966..6022
                     /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     6023..6047
                     /label=TLP12_p6/7-mCherry_F_73
     primer_bind     6025..6047
                     /label=TLP21_p13/14-mCherry_F_63
     misc_feature    6025..6028
                     /label=p13/14-connector
     CDS             join(6029..6736,6737..6757,6758..6829)
                     /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."
     misc_feature    6029..6736
                     /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(6208..6227)
                     /label=MMS123_mCherry_180R
     primer_bind     6615..6634
                     /label=MMS122_mCherry_600F
     primer_bind     complement(6652..6671)
                     /label=MMS124_mCherry_620R
     primer_bind     complement(6724..6736)
                     /label=TLP9_p5/6-NotI_R_66
     CDS             6737..6757
                     /locus_tag="Linker"
                     /label=Linker
     misc_signal     6758..6809
                     /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     6810..6829
                     /locus_tag="NLS"
                     /label=NLS
     primer_bind     complement(6827..6850)
                     /label=MMC425_CAG-backbone_EMMA-p14_R
     misc_feature    6847..6850
                     /locus_tag="p14-15 connector"
                     /label=p14-15 connector
     polyA_site      6960..7011
                     /locus_tag="SPA"
                     /label=SPA
     misc_feature    7012..7132
                     /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(7056..7074)
                     /label=TLS15_C2MAZ_R_60
     misc_feature    7140..7143
                     /label=p15/16-connector
     misc_feature    7144..8357
                     /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    8358..8361
                     /label=p25/V-connector
     terminator      complement(8435..8464)
                     /locus_tag="T3Te terminator"
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     primer_bind     complement(8450..8469)
                     /label=MMS150_EMMA_Receiver_2
     rep_origin      complement(8486..9073)
                     /direction=LEFT
                     /locus_tag="ori"
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8549..8568)
                     /label=MMS_EMMA_Reverse_New
     terminator      complement(9085..9112)
                     /locus_tag="T7Te terminator"
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     9085..9099
                     /label=MMS150_EMMA_Receiver_2
     CDS             complement(9136..9996)
                     /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    9576..9581
                     /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(9997..10099)
                     /locus_tag="cat promoter"
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     primer_bind     10084..10103
                     /label=MMS149_EMMA_Receiver_1
     terminator      complement(10100..10131)
                     /locus_tag="tonB terminator"
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription
                     terminator"
ORIGIN
        1 aatggtcagt attgagcgat atctagagaa ttcgtatagg gcgttaatta actagagccc
       61 catcctcact gactccgtcc tggagttgga tgagagataa tggccttacg ttgtgccagg
      121 ggagggtcgg gctggattta gcaagattta ccttctccaa agagcggtgc tgcagtggca
      181 cagctgccca cggaggtggg ggggtcaccg tccctggagg tgatgaagaa ctgtggggat
      241 gtggcactga gggacatggc cagtgggcac ggtgggtggg ttggggttgg tcttggggat
      301 cttggagggc ttttccagcc ttcatgattt gacgattgta tgaacatcta catggcaatt
      361 ctccagctgc ctgtcccagt cctactgacc cagctgtatc tctccaggca agctcttcca
      421 ccccttctgc ttgcatccag acaccatcaa acatgcaggc tcagacacag ggaccagcag
      481 tgtctgtggc ctttttgtgc tcctctccat gctgggtttt aacttgctct ttgtccttct
      541 atcctatctt cttatcctta aggctgttct gaacgctgtg acttggagag tgtcccagag
      601 ccctcaacac ctgcatgtcc cacgtccatg ctgtcctgca cttccttatc cccaagatct
      661 gcctctccgt gatgcactga attggcaaac atgtgtcacc ccagaccaac aatgtcacag
      721 caaactcccc cttgatagga caagggggaa tggctttaca ctgagacagg ggaggtttgg
      781 gttggatatg aggaggcagt ttttccccca gagggtggtg acgcactgaa caggttgccc
      841 aaggaggctg tggatgcccc atccctgcag gcattcaagg ccaggctgga tgtggctctg
      901 ggcagcctgg gctgctggtt gatgaccctg cacatagcag ggggttggat ctggatgagc
      961 actgtgctcc tttgcaaccc aggccgttct atgattctgt cattctaaat ctctctttca
     1021 gcctaaagct ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag
     1081 cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg
     1141 ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct
     1201 gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc
     1261 tctagggacg ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     1321 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     1381 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     1441 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     1501 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     1561 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     1621 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     1681 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     1741 gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg
     1801 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
     1861 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
     1921 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
     1981 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
     2041 tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt
     2101 gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt
     2161 gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg
     2221 cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt
     2281 gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg
     2341 ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct gcacccccct
     2401 ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg
     2461 gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg
     2521 cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt
     2581 cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga
     2641 cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct
     2701 agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     2761 cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg
     2821 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     2881 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     2941 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattcctcga ctccgcctag
     3001 gcgcgccacc tcgaggccgc caccatgaag tgggtgacct tcatcagcct gctgtttctg
     3061 ttcagcagcg cctactctag agggggcgga gcaagcaaag gcgagaggct gtttagaggg
     3121 aaagtgccaa tcctggtgga actgaaagga gacgtgaacg gccacaagtt ctctgtccga
     3181 ggcaagggga aaggagatgc cacacgggga aaactgactc tgaagtttat ctgcaccaca
     3241 ggcaagctgc ccgtgccttg gccaaccctg gtcactaccc tgacatacgg cgtgcagtgt
     3301 ttcagccgat atccaaaaca catgaagcgg catgacttct ttaaaagtgc tatgcccaag
     3361 ggctacgtgc aggagcgcac catttcattc aagaaggatg ggaagtacaa gacccgggcc
     3421 gaggtgaagt ttgaagggag aaccctggtc aaccgcatca agctgaaagg acgcgacttc
     3481 aaggaaaaag ggaatattct gggacacaaa ctgagataca acttcaacag ccataaggtg
     3541 tacatcacag cagataagag aaaaaacggc atcaaggcca aattcaagat taggcacaat
     3601 gtgaaggacg gctccgtcca gctggctgat cattaccagc agaacacacc tattggcaga
     3661 gggcccgtgc tgctgcctag gaatcattat ctgagcactc gctccaaact gtctaaggac
     3721 cccaaagaga agcgggatca catggtgctg ctggaatttg tcaccgccgc tggcatcaaa
     3781 catgggcggg acgagagata caagggcggc agcggcggca ccagcggcgg cggcggcggc
     3841 agcggcgcag aaatcggtac tggctttcca ttcgaccccc attatgtgga agtcctgggc
     3901 gagcgcatgc actacgtcga tgttggtccg cgcgatggca cccctgtgct gttcctgcac
     3961 ggtaacccga cctcctccta cgtgtggcgc aacatcatcc cgcatgttgc accgacccat
     4021 cgctgcattg ctccagacct gatcggtatg ggcaaatccg acaaaccaga cctgggttat
     4081 ttcttcgacg accacgtccg cttcatggat gccttcatcg aagccctggg tctggaagag
     4141 gtcgtcctgg tcattcacga ctggggctcc gctctgggtt tccactgggc caagcgcaat
     4201 ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc gccctatccc gacctgggac
     4261 gaatggccag aatttgcccg cgagaccttc caggccttcc gcaccaccga cgtcggccgc
     4321 aagctgatca tcgatcagaa cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc
     4381 ccgctgactg aagtcgagat ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc
     4441 gagccactgt ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc
     4501 gcgctggtcg aagaatacat ggactggctg caccagtccc ctgtcccgaa gctgctgttc
     4561 tggggcaccc caggcgttct gatcccaccg gccgaagccg ctcgcctggc caaaagcctg
     4621 cctaactgca aggctgtgga catcggcccg ggtctgaatc tgctgcaaga agacaacccg
     4681 gacctgatcg gcagcgagat cgcgcgctgg ctgtcgacgc tggagatttc cggctaagcg
     4741 gccgcggtac cgctcgataa gcttgatatc gaattccgcc cccccccctc tccctccccc
     4801 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     4861 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     4921 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     4981 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     5041 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     5101 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     5161 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     5221 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     5281 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     5341 gaaaaacacg atgataatcg tcatgaccga gtacaagccc acggtgcgcc tcgccacccg
     5401 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     5461 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     5521 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     5581 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     5641 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     5701 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggtg tgtcgcccga
     5761 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     5821 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     5881 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     5941 gacccgcaag cccggtgcct cacttgccac aaacttctct ctgctaaagc aagcaggtga
     6001 tgttgaagaa aaccccgggc ctgcctacat ggtgagcaag ggcgaggagg ataacatggc
     6061 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     6121 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     6181 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     6241 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     6301 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     6361 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     6421 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     6481 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     6541 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     6601 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     6661 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     6721 ggacgagctg tacaagacct caggcaggac aggtagtcca aaaaagaaga gaaaggtaga
     6781 tccaaaaaag aagagaaagg tagatccaaa aaagaagaga aaggtattat aagcggccgc
     6841 tcgatagcaa atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc
     6901 gaccgaaagg agcgcacgac cccatgcatc gatgatctag aactagtgga tccccgatcc
     6961 aataaaagat ctttattttc attagatctg tgtgttggtt ttttgtgtgg gcttggggga
     7021 gggggaggcc agaatgactc caagagctac aggaaggcag gtcagagacc ccactggaca
     7081 aacagtggct ggactctgca ccataacaca caatcaacag gggagtgagc tggatccggc
     7141 cctctagagc cccatcctca ctgactccgt cctggagttg gatgagagat aatggcctta
     7201 cgttgtgcca ggggagggtc gggctggatt tagcaagatt taccttctcc aaagagcggt
     7261 gctgcagtgg cacagctgcc cacggaggtg ggggggtcac cgtccctgga ggtgatgaag
     7321 aactgtgggg atgtggcact gagggacatg gccagtgggc acggtgggtg ggttggggtt
     7381 ggtcttgggg atcttggagg gcttttccag ccttcatgat ttgacgattg tatgaacatc
     7441 tacatggcaa ttctccagct gcctgtccca gtcctactga cccagctgta tctctccagg
     7501 caagctcttc caccccttct gcttgcatcc agacaccatc aaacatgcag gctcagacac
     7561 agggaccagc agtgtctgtg gcctttttgt gctcctctcc atgctgggtt ttaacttgct
     7621 ctttgtcctt ctatcctatc ttcttatcct taaggctgtt ctgaacgctg tgacttggag
     7681 agtgtcccag agccctcaac acctgcatgt cccacgtcca tgctgtcctg cacttcctta
     7741 tccccaagat ctgcctctcc gtgatgcact gaattggcaa acatgtgtca ccccagacca
     7801 acaatgtcac agcaaactcc cccttgatag gacaaggggg aatggcttta cactgagaca
     7861 ggggaggttt gggttggata tgaggaggca gtttttcccc cagagggtgg tgacgcactg
     7921 aacaggttgc ccaaggaggc tgtggatgcc ccatccctgc aggcattcaa ggccaggctg
     7981 gatgtggctc tgggcagcct gggctgctgg ttgatgaccc tgcacatagc agggggttgg
     8041 atctggatga gcactgtgct cctttgcaac ccaggccgtt ctatgattct gtcattctaa
     8101 atctctcttt cagcctaaag ctttttcccc gtatcccccc aggtgtctgc aggctcaaag
     8161 agcagcgaga agcgttcaga ggaaagcgat cccgtgccac cttccccgtg cccgggctgt
     8221 ccccgcacgc tgccggctcg gggatgcggg gggagcgccg gaccggagcg gagccccggg
     8281 cggctcgctg ctgcccccta gcgggggagg gacgtaatta catccctggg ggctttgggg
     8341 gggggctgtc cctctagacg ataacgaatt caagcttgat atcattcagg acgagcctca
     8401 gactccagcg taactggact gcaatcaact cactggctca ccttcacggg tgggcctttc
     8461 ttcggtagaa aatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     8521 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     8581 ctctttttcc gaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     8641 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     8701 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     8761 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     8821 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     8881 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     8941 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     9001 tgtcgggttt cgccacctct gacttgagca tcgatttttg tgatgctcgt caggggggcg
     9061 gagcctatgg aaaaacgcca gcaacgcaga aaggcccacc cgaaggtgag ccaggtgatt
     9121 acatttgggc cctcattacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     9181 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tgcgggaggg
     9241 cttaccatct ggccccagtg ctgcaatgat accgcgagaa ccacgctcac cggctccaga
     9301 tttatcagca ataaaccagc cagccgggag ggccgagcgc agaagtgatc ctgcaacttt
     9361 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     9421 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     9481 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     9541 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgccagaa gtaagttggc
     9601 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     9661 cgtgagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     9721 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     9781 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgtaaactct caaggatctt
     9841 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     9901 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     9961 gggaataagg gcgacacgga aatgttgaat actcatttta gcttccttag ctcctgaaaa
    10021 tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga
    10081 acctcttacg tgccgatcaa gtcaaaagcc tccggtcgga ggcttttgac tttctgctat
    10141 ggaggtcagg tatgattta
//
