LOCUS       Exported                9219 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Express gRNA and Cas9 in Leishmania with Neomycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    pLdCN
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9219)
  AUTHORS   Zhang WW, Lypaczewski P, Matlashewski G
  TITLE     Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To 
            Target a Multigene Family, Induce Chromosomal Translocation, and 
            Study DNA Break Repair Mechanisms.
  JOURNAL   mSphere. 2017 Jan 18;2(1). pii: e00340-16. doi: 
            10.1128/mSphere.00340-16. eCollection 2017 Jan-Feb.
  PUBMED    28124028
REFERENCE   2  (bases 1 to 9219)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9219
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_RNA        60..135
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      377..386
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             386..451
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             458..478
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             503..4603
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4604..4651
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4679..4696)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             5888..6682
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHKAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5942..5961)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6552..6571
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6708..6727)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6709..6727)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6814..6831)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6985..7573)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7065..7084)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7744..8604)
                     /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"
     primer_bind     8367..8386
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8605..8709)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8777..8795
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8833..8855)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8955..8974
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        9055..9073
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     9055..9072
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 ttatcggggg ctcggttgag tttttggttt ggtgattttg tgggtcttcg agaagacctg
       61 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
      121 gcaccgagtc ggtgcttttt tggccggcat ggtcccagcc tcctcgctgg cgccggctgg
      181 gcaacatgct tcggcatggc gaatgggacg gatccccggg taccgagctc tctcttctcc
      241 ttcccctctc tctcctctct cctctctcca gagctctctc ttctccttcc cctctctctc
      301 ctctctcctc tctccagagc tcgaattcgc aagcttagca cctgcctgaa atcacttttt
      361 ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact acaaggatca
      421 tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg
      481 tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa
      541 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
      601 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
      661 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag
      721 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
      781 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
      841 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
      901 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
      961 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
     1021 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     1081 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     1141 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
     1201 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
     1261 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga
     1321 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
     1381 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
     1441 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct
     1501 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
     1561 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
     1621 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
     1681 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat
     1741 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
     1801 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
     1861 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
     1921 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
     1981 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
     2041 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
     2101 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa
     2161 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
     2221 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
     2281 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
     2341 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
     2401 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
     2461 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca
     2521 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat
     2581 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc
     2641 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa
     2701 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca
     2761 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca
     2821 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca
     2881 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta
     2941 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc
     3001 cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa
     3061 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga
     3121 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca
     3181 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc
     3241 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat
     3301 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa
     3361 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa
     3421 agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg
     3481 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa
     3541 ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc
     3601 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa
     3661 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg
     3721 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat
     3781 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag
     3841 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt
     3901 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc
     3961 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt
     4021 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct
     4081 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct
     4141 ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa
     4201 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca
     4261 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag
     4321 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta
     4381 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac
     4441 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa
     4501 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg
     4561 cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac
     4621 gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc ctagagctcg ctgatcagcc
     4681 tcgactgtgc cttctagttg ccagccatct gttggatccg cggccgcctc gagcccgggc
     4741 gccgtcgacc gctgcttgcg ttcgcatgga cgccacgttc gacccgatcg ggcagcaggc
     4801 cgcggagacg cggcagcgca aacgtgcccg gcgggtggct cgttccggag gaggcgttcc
     4861 gccgtcgcta cgtcagcatg caagaggatc attgctttgc ggttgaggtg gcggagctca
     4921 actaccagcg tgccgtggaa tacgttcatc aagcgtcagc cgccggcgcg gtcgcgacac
     4981 ctgctcggcg gagcggccac tgtgctgctc tcttgacggc cggtcgcgac gcagggccgc
     5041 tggtgggtct ccagcgggta cggtgcgatc aggcagccat gacggctacg ctgagcacgc
     5101 ttgcgaagga gctggctgca ctgtcgccgt agcgcgatgc gagacgtatc gagcgcatgc
     5161 cagtggggtg ctgggcaaca cttggcggcg cgtggctctt tctcactggt ttgggcacgc
     5221 ccacgctgtg ggtgtgtcgg tgtctgccct gccgctctct ctcctgatct ctcgtcctgg
     5281 caacaacaac acaccgcgaa tgagttgccc acaagccatc tgcagtccgc acgcgcgtcc
     5341 gggccttgcg gtgtggatgc gagcatcgcg cattgccggc caccgccgcg tgccaaaagc
     5401 acacacatcg ctccccctca cgtggtgagc gtctgcgcgt gtgtgtctgt gtgcataaat
     5461 gcgtacgtgt gcgctcggtc cacggtcacc atcgcgcccc gcggcatcgg tgctcgcacc
     5521 tttccattct ctccgtacgg cgttcgccac tctgctaact cttctcccct ttcctctctc
     5581 cgcatctgcg ccttgtgtgc cgtgtgtgcg ccccacacgt actctacacg ccttctctgc
     5641 atgcgctcct cgcctgcctg aaacgatgac gtgcgccgcc actcgctcac ccgctcccat
     5701 ccacatgcat tcctgcaccc tacagcgact ctaagccgta cattccatct cctcaacacc
     5761 cactcgcttc cttctgctct cacctattac ttcgccagcc cacatatctg ctataacctg
     5821 ccctccccca cccgcttccc acacatccgc caccgctacg aagcctcggt accgagctcg
     5881 aattcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     5941 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     6001 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     6061 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     6121 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     6181 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     6241 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc acaaagcgaa
     6301 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     6361 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     6421 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     6481 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     6541 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     6601 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     6661 ccttcttgac gagttcttct gatcgatgat atcagatctg ccggtctccc tatagtgagt
     6721 cgtattaatt tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag
     6781 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6841 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6901 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6961 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     7021 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     7081 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     7141 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     7201 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     7261 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     7321 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     7381 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     7441 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     7501 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7561 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     7621 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7681 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     7741 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7801 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7861 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7921 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7981 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     8041 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     8101 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     8161 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     8221 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     8281 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     8341 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     8401 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     8461 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     8521 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8581 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     8641 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     8701 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     8761 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
     8821 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     8881 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     8941 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tggacatatt
     9001 gtcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag
     9061 gtgacactat agaactcgag tgtgagttat gaggtctgcg attgacgtag gagttgcaag
     9121 ggggaggggg tatgaacggg gtgggtagag cttttttagg tggaagtagt gagagggtgg
     9181 gcttgtgaga atttgaggtg tgttcgtgat gtgtggatc
//
