LOCUS       sequence-450825-        9923 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CBh-eSpCas9(1.1)-2A-GFP, and 2X hU6-sgRNA (Sp) with BbsI golden gate
            cloning backbone dual gRNAs. sgRNA uses 3TC scaffold for increased 
            sgRNA expression. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9923)
  AUTHORS   Chey YCJ, Gierus L, Lushington C, Arudkumar JC, B Geiger A, Staker 
            LG, Robertson LJ, Pfitzner C, Kennedy JG, Lee RHB, Godahewa GI, 
            Adikusuma F, Thomas PQ
  TITLE     Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA 
            transcript levels through scaffold poly-T tract reduction.
  JOURNAL   BMC Genomics. 2025 Feb 12;26(1):138. doi: 
            10.1186/s12864-025-11317-2.
  PUBMED    39939860
REFERENCE   2  (bases 1 to 9923)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC 
            Genomics."; date: "2025-02-12"; volume: "26(1):138. doi"; pages: " 
            10.1186/s12864-025-11317-2"
COMMENT     Sequence Label: PDG458 eSp(1.1) V3
FEATURES             Location/Qualifiers
     source          1..9923
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          157..384
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      396..405
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             405..470
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             477..497
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(522..3059,3060..3062,3063..3524,3525..3527,3528..3695,
                     3696..3698,3699..4622)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4623..4670
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4686..4739
                     /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"
     CDS             join(4740..4742,4743..5453)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4740..4742,4743..5453)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5043..5062)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5390..5411
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5481..5498)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5487..5694
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(5772..5847)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5865..6113)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     rep_origin      6196..6651
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6283..6302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6493..6514
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6668..6687)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6787..6809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6847..6865)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6933..7037
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7038..7106,7107..7898)
                     /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     complement(7256..7275)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8069..8657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8558..8577
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8719..8967
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8986..9061
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        9158..9443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ggggctggga grggcggggc ggggcgaggg gncggggcgg ggncgaggcg gagaggtgcg
       61 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      121 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      181 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      241 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
      301 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
      361 ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg
      421 acggagacta caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa
      481 agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg
      541 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
      601 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
      661 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
      721 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
      781 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
      841 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
      901 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
      961 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     1021 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     1081 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     1141 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     1201 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     1261 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     1321 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     1381 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     1441 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     1501 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     1561 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     1621 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     1681 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     1741 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     1801 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     1861 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     1921 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     1981 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     2041 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     2101 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     2161 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     2221 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     2281 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     2341 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     2401 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     2461 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     2521 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     2581 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     2641 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     2701 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     2761 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     2821 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     2881 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     2941 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     3001 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctgg
     3061 ccgacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     3121 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     3181 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     3241 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     3301 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     3361 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     3421 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     3481 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctgcgctg gaaagcgagt
     3541 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     3601 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     3661 ccgagattac cctggccaac ggcgagatcc ggaaggcgcc tctgatcgag acaaacggcg
     3721 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     3781 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     3841 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     3901 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     3961 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     4021 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     4081 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     4141 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     4201 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     4261 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     4321 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     4381 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     4441 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     4501 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     4561 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     4621 acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag gaattcggca
     4681 gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag
     4741 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     4801 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     4861 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     4921 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     4981 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     5041 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     5101 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     5161 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     5221 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     5281 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     5341 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     5401 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggaattct
     5461 aactagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     5521 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     5581 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     5641 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     5701 gcaattggcg cacgcgctaa aaacggacta gccttatttt aacttgctat ttctagctct
     5761 aaaacaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt
     5821 tcaacttgct atttctagct ctgaaacgtc ttctcgaaga ccccggtgtt tcgtcctttc
     5881 cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag
     5941 tccattttaa aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt
     6001 cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattccaat tatctctcta
     6061 acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctccctgcag
     6121 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     6181 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     6241 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     6301 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     6361 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     6421 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     6481 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     6541 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     6601 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     6661 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     6721 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     6781 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     6841 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     6901 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     6961 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     7021 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     7081 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     7141 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     7201 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     7261 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     7321 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     7381 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     7441 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     7501 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     7561 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     7621 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     7681 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     7741 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     7801 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     7861 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     7921 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     7981 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     8041 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     8101 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     8161 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     8221 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     8281 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     8341 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     8401 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     8461 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     8521 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     8581 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     8641 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     8701 ggccttttgc tcacatgtga gggcctattt cccatgattc cttcatattt gcatatacga
     8761 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
     8821 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     8881 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     8941 tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa gacctgtttc agagctagaa
     9001 atagcaagtt gaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     9061 cttttttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg
     9121 tgcgccaatt ctgcagacaa atggctctag aggtacccgt tacataactt acggtaaatg
     9181 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag
     9241 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     9301 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     9361 cctggcattg tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     9421 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
     9481 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     9541 cgatgggggc gggggggggg ggggggnngc gcgncncagg gcggggnggg ggnnggcggg
     9601 ggggkggggg gsggngnggs gggggggggg gggggggsgk kkyyataaca tcwksmcccc
     9661 cctcttccac ccaccatgat caacgataag agtggggggt gacgtaagta ttgatagaag
     9721 gggcgtcctt attcacagtc gccgggtcgc cccccccccc tgcttcacct cccccccccc
     9781 cccccccccc ccccccccta tttttttttt tttttttttt tatttttttg gggcggggag
     9841 gggggggggg gggggggggg ggggcgcgcc aggcgggggg gggcggggcg aggggcgggg
     9901 cggggcgagg cggagaggtg cgg
//
