LOCUS       pLenti6_cppt-CMV       10240 bp DNA     circular SYN 28-JUN-2024
DEFINITION  pLEN6-stabl.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10240)
  AUTHORS   Tamina Lebek
  TITLE     Direct Submission
  JOURNAL   Exported Jul 2, 2024 from SnapGene 7.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1058..1284
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1285..1465
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1512..1637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2130..2363
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2548..2592
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2888..3005
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        3083..3388
                     /locus_tag="CMV enhancer"
                     /label=CMV enhancer
     enhancer        3084..3387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3388..3591
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    3713..3721
                     /label=kozak
     CDS             join(3722..3775,3776..3781,3782..4501,4502..4543,
                     4544..5248)
                     /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"
     regulatory      4796..4805
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5252..5263
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5255..5308
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     misc_feature    5315..5323
                     /label=kozak
     regulatory      5318..5327
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5324..6031,6032..6052,6053..6127)
                     /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    5324..6031
                     /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). 
                     "
     CDS             6032..6052
                     /locus_tag="Linker"
                     /label=Linker
     misc_signal     6053..6104
                     /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."
     CDS             6053..6073
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6077..6097
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6101..6121
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_signal     6105..6124
                     /locus_tag="NLS"
                     /label=NLS
     promoter        6752..7251
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     promoter        7265..7312
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7331..7729
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             7816..8049
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8121..8242
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    8127..8181
                     /locus_tag="SV40pA"
                     /label=SV40pA
     polyA_site      8177..8223
                     /locus_tag="SV40pA core"
                     /label=SV40pA core
     rep_origin      8282..8417
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(8438..8456)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8466..8482)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      8624..9079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        9105..9209
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9210..9278,9279..10070)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
     1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
     1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
     1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2761 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     2821 attcgattag tgaacggatc tcgacggtat cgatgtcgac agtacaaatg gcagtattca
     2881 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     2941 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     3001 attttcgggt ttattacagg gacagcagag atccagtttg gaaagggcta gcgactgggt
     3061 ctgcgataag cttgggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     3121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     3181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     3241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     3301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     3361 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     3421 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     3481 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     3541 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg
     3601 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccga
     3661 ctctagagga tctgttaaca tatgaattcg ctagcatgca tggatcctcg aggccgccac
     3721 catgaagtgg gtgaccttca tcagcctgct gtttctgttc agcagcgcct actctagagg
     3781 gggcggagca agcaaaggcg agaggctgtt tagagggaaa gtgccaatcc tggtggaact
     3841 gaaaggagac gtgaacggcc acaagttctc tgtccgaggc aaggggaaag gagatgccac
     3901 acggggaaaa ctgactctga agtttatctg caccacaggc aagctgcccg tgccttggcc
     3961 aaccctggtc actaccctga catacggcgt gcagtgtttc agccgatatc caaaacacat
     4021 gaagcggcat gacttcttta aaagtgctat gcccaagggc tacgtgcagg agcgcaccat
     4081 ttcattcaag aaggatggga agtacaagac ccgggccgag gtgaagtttg aagggagaac
     4141 cctggtcaac cgcatcaagc tgaaaggacg cgacttcaag gaaaaaggga atattctggg
     4201 acacaaactg agatacaact tcaacagcca taaggtgtac atcacagcag ataagagaaa
     4261 aaacggcatc aaggccaaat tcaagattag gcacaatgtg aaggacggct ccgtccagct
     4321 ggctgatcat taccagcaga acacacctat tggcagaggg cccgtgctgc tgcctaggaa
     4381 tcattatctg agcactcgct ccaaactgtc taaggacccc aaagagaagc gggatcacat
     4441 ggtgctgctg gaatttgtca ccgccgctgg catcaaacat gggcgggacg agagatacaa
     4501 gggcggcagc ggcggcacca gcggcggcgg cggcggcagc ggcgtgagca agggcgagga
     4561 ggataacatg gcctctctcc cagcgacaca tgagttacac atctttggct ccatcaacgg
     4621 tgtggacttt gacatggtgg gtcagggcac cggcaatcca aatgatggtt atgaggagtt
     4681 aaacctgaag tccaccaagg gtgacctcca gttctccccc tggattctgg tccctcatat
     4741 cgggtatggc ttccatcagt acctgcccta ccctgacggg atgtcgcctt tccaggccgc
     4801 catggtagat ggctccggat accaagtcca tcgcacaatg cagtttgaag atggtgcctc
     4861 ccttactgtt aactaccgct acacctacga gggaagccac atcaaaggag aggcccaggt
     4921 gaaggggact ggtttccctg ctgacggtcc tgtgatgacc aactcgctga ccgctgcgga
     4981 ctggtgcagg tcgaagaaga cttaccccaa cgacaaaacc atcatcagta cctttaagtg
     5041 gagttacacc actggaaatg gcaagcgcta ccggagcact gcgcggacca cctacacctt
     5101 tgccaagcca atggcggcta actatctgaa gaaccagccg atgtacgtgt tccgtaagac
     5161 ggagctcaag cactccaaga ccgagctcaa cttcaaggag tggcaaaagg cctttaccga
     5221 tgtgatgggc atggacgagc tgtacaaggg tatagagggc agaggaagtc ttctaacatg
     5281 cggtgacgtg gaggagaatc ccggccctgg cgtcgccgcc accatggtga gcaagggcga
     5341 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     5401 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     5461 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     5521 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     5581 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     5641 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     5701 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     5761 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     5821 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     5881 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     5941 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     6001 ctccaccggc ggcatggacg agctgtacaa gacctcaggc aggacaggta gtccaaaaaa
     6061 gaagagaaag gtagatccaa aaaagaagag aaaggtagat ccaaaaaaga agagaaaggt
     6121 attataagcg gcccgagcat cttaccgcca tttattccca tatttgttct gtttttcttg
     6181 atttgggtat acatttaaat gttaataaaa caaaatggtg gggcaatcat ttacattttt
     6241 agggatatgt aattactagt tcaggtgtat tgccacaaga caaacatgtt aagaaacttt
     6301 cccgttattt acgctctgtt cctgttaatc aacctctgga ttacaaaatt tgtgaaagat
     6361 tgactgatat tcttaactat gttgctcctt ttacgctgtg tggatatgct gctttatagc
     6421 ctctgtatct agctattgct tcccgtacgg ctttcgtttt ctcctccttg tataaatcct
     6481 ggttgctgtc tcttttagag gagttgtggc ccgttgtccg tcaacgtggc gtggtgtgct
     6541 ctgtgtttgc tgacgcaacc cccactggct ggggcattgc caccacctgt caactccttt
     6601 ctgggacttt cgctttcccc ctcccgatcg ccacggcaga actcatcgcc gcctgccttg
     6661 cccgctgctg gacaggggct aggttgctgg gcactgataa ttccgtggtg tgtcgaacgc
     6721 gtaccggtta gtaatgacga tggtacctac cgggtagggg aggcgctttt cccaaggcag
     6781 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
     6841 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
     6901 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
     6961 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc
     7021 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
     7081 ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg
     7141 ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa
     7201 aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gactctagac
     7261 acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     7321 gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
     7381 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
     7441 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     7501 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     7561 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     7621 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     7681 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacaattcga
     7741 gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     7801 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     7861 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     7921 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     7981 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     8041 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     8101 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     8161 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     8221 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     8281 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     8341 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     8401 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     8461 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     8521 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     8581 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     8641 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     8701 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     8761 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     8821 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     8881 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     8941 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     9001 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     9061 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     9121 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     9181 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     9241 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     9301 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     9361 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     9421 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     9481 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     9541 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     9601 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     9661 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     9721 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     9781 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     9841 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     9901 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     9961 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
    10021 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
    10081 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
    10141 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
    10201 actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
