LOCUS       sequence-450777-        9292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CBh-SpCas9-2A-GFP, and hU6-sgRNA (Sp) with a BbsI golden gate 
            cloning backbone for sgRNA. sgRNA uses 3TC scaffold for increased 
            sgRNA expression. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9292)
  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 9292)
  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: PX458 V3
FEATURES             Location/Qualifiers
     source          1..9292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(4081..4101)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4108..4173)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      4173..4182
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          4194..4421
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     enhancer        4705..4990
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     misc_RNA        complement(5087..5162)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5181..5429)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5239..5258)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5409..5429)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(5491..6079)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5571..5590)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6250..7041,7042..7110))
                     /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     6873..6892
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7111..7215)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7283..7301
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7339..7361)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7461..7480
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(7497..7952)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7634..7655)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7846..7865
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   8027..8167
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8038..8167
                     /label=AAV2 ITR
     polyA_signal    8176..8383
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     8372..8389
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(8417..9127,9128..9130))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(8417..9127,9128..9130))
                     /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(8459..8480)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     8808..8827
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             complement(9131..9184)
                     /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             complement(9200..9247)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(9248..9292,1..4056))
                     /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             complement(join(9248..9292,1..4056))
                     /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"
ORIGIN
        1 ggtgatgctc tggtggatca gggtggcgtc cagcacctct ttggtgctgg tgtacctctt
       61 ccggtcgatg gtggtgtcaa agtacttgaa ggcggcaggg gctcccagat tggtcagggt
      121 aaacaggtgg atgatattct cggcctgctc tctgatgggc ttatcccggt gcttgttgta
      181 ggcggacagc actttgtcca gattagcgtc ggccaggatc actctcttgg agaactcgct
      241 gatctgctcg atgatctcgt ccaggtagtg cttgtgctgt tccacaaaca gctgtttctg
      301 ctcattatcc tcgggggagc ccttcagctt ctcatagtgg ctggccaggt acaggaagtt
      361 cacatatttg gagggcaggg ccagttcgtt tcccttctgc agttcgccgg cagaggccag
      421 cattctcttc cggccgtttt ccagctcgaa cagggagtac ttaggcagct tgatgatcag
      481 gtcctttttc acttctttgt agcccttggc ttccagaaag tcgatgggat tcttctcgaa
      541 gctgcttctt tccatgatgg tgatccccag cagctctttc acactcttca gtttcttgga
      601 cttgcccttt tccactttgg ccaccaccag cacagaatag gccacggtgg ggctgtcgaa
      661 gccgccgtac ttcttagggt cccagtcctt ctttctggcg atcagcttat cgctgttcct
      721 cttgggcagg atagactctt tgctgaagcc gcctgtctgc acctcggtct ttttcacgat
      781 attcacttgg ggcatgctca gcactttccg cacggtggca aaatcccggc ccttatccca
      841 cacgatctcc ccggtttcgc cgtttgtctc gatcagaggc cgcttccgga tctcgccgtt
      901 ggccagggta atctcggtct tgaaaaagtt catgatgttg ctgtagaaga agtacttggc
      961 ggtagccttg ccgatttcct gctcgctctt ggcgatcatc ttccgcacgt cgtacacctt
     1021 gtagtcgccg tacacgaact cgctttccag cttagggtac tttttgatca gggcggttcc
     1081 cacgacggcg ttcaggtagg cgtcgtgggc gtggtggtag ttgttgatct cgcgcacttt
     1141 gtaaaactgg aaatccttcc ggaaatcgga caccagcttg gacttcaggg tgatcacttt
     1201 cacttcccgg atcagcttgt cattctcgtc gtacttagtg ttcatccggg agtccaggat
     1261 ctgtgccacg tgctttgtga tctgccgggt ttccaccagc tgtctcttga tgaagccggc
     1321 cttatccagt tcgctcaggc cgcctctctc ggccttggtc agattgtcga actttctctg
     1381 ggtaatcagc ttggcgttca gcagctgccg ccagtagttc ttcatcttct tcacgacctc
     1441 ttcggagggc acgttgtcgc tcttgccccg gttcttgtcg cttctggtca gcaccttgtt
     1501 gtcgatggag tcgtccttca gaaagctctg aggcacgata tggtccacat cgtagtcgga
     1561 cagccggttg atgtccagtt cctggtccac gtacatatcc cgcccattct gcaggtagta
     1621 caggtacagc ttctcgttct gcagctgggt gttttccacg gggtgttctt tcaggatctg
     1681 gctgcccagc tctttgatgc cctcttcgat ccgcttcatt ctctcgcggc tgttcttctg
     1741 tcccttctgg gtggtctggt tctctctggc catttcgatc acgatgttct cgggcttgtg
     1801 ccggcccatc actttcacga gctcgtccac caccttcact gtctgcagga tgcccttctt
     1861 aatggcgggg ctgccggcca gattggcaat gtgctcgtgc aggctatcgc cctggccgga
     1921 cacctgggct ttctggatgt cctctttaaa ggtcaggctg tcgtcgtgga tcagctgcat
     1981 gaagtttctg ttggcgaagc cgtcggactt caggaaatcc aggattgtct tgccggactg
     2041 cttgtcccgg atgccgttga tcagcttccg gctcagcctg ccccagccgg tgtatctccg
     2101 ccgcttcagc tgcttcatca ctttgtcgtc gaacaggtgg gcataggttt tcagccgttc
     2161 ctcgatcatc tctctgtcct caaacagtgt cagggtcagc acgatatctt ccagaatgtc
     2221 ctcgttttcc tcattgtcca ggaagtcctt gtccttgata attttcagca gatcgtggta
     2281 tgtgcccagg gaggcgttga accgatcttc cacgccggag atttccacgg agtcgaagca
     2341 ctcgattttc ttgaagtagt cctctttcag ctgcttcacg gtcactttcc ggttggtctt
     2401 gaacagcagg tccacgatgg cctttttctg ctcgccgctc aggaaggcgg gctttctcat
     2461 tccctcggtc acgtatttca ctttggtcag ctcgttatac acggtgaagt actcgtacag
     2521 caggctgtgc ttgggcagca ccttctcgtt gggcaggttc ttatcgaagt tggtcatccg
     2581 ctcgatgaag ctctgggcgg aagcgccctt gtccaccact tcctcgaagt tccagggggt
     2641 gatggtttcc tcgctctttc tggtcatcca ggcgaatctg ctgtttcccc tggccagagg
     2701 gcccacgtag taggggatgc ggaaggtcag gatcttctcg atcttttccc ggttgtcctt
     2761 caggaatggg taaaaatctt cctgccgccg cagaatggcg tgcagctctc ccaggtggat
     2821 ctggtggggg atgctgccgt tgtcgaaggt ccgctgcttc cgcagcaggt cctctctgtt
     2881 cagcttcacg agcagttcct cggtgccgtc catcttttcc aggatgggct tgatgaactt
     2941 gtagaactct tcctggctgg ctccgccgtc aatgtagccg gcgtagccgt tcttgctctg
     3001 gtcgaagaaa atctctttgt acttctcagg cagctgctgc cgcacgagag ctttcagcag
     3061 ggtcaggtcc tggtggtgct cgtcgtatct cttgatcata gaggcgctca ggggggcctt
     3121 ggtgatctcg gtgttcactc tcaggatgtc gctcagcagg atggcgtcgg acaggttctt
     3181 ggcggccaga aacaggtcgg cgtactggtc gccgatctgg gccagcaggt tgtccaggtc
     3241 gtcgtcgtag gtgtccttgc tcagctgcag tttggcatcc tcggccaggt cgaagttgct
     3301 cttgaagttg ggggtcaggc ccaggctcag ggcaatcagg tttccgaaca ggccattctt
     3361 cttctcgccg ggcagctggg cgatcagatt ttccagccgt ctgctcttgc tcagtctggc
     3421 agacaggatg gccttggcgt ccacgccgct ggcgttgatg gggttttcct cgaacagctg
     3481 gttgtaggtc tgcaccagct ggatgaacag cttgtccacg tcgctgttgt cggggttcag
     3541 gtcgccctcg atcaggaagt ggccccggaa cttgatcatg tgggccaggg ccagatagat
     3601 cagccgcagg tcggccttgt cggtgctgtc caccagtttc tttctcaggt ggtagatggt
     3661 ggggtacttc tcgtggtagg ccacctcgtc cacgatgttg ccgaagatgg ggtgccgctc
     3721 gtgcttctta tcctcttcca ccaggaagga ctcttccagt ctgtggaaga agctgtcgtc
     3781 caccttggcc atctcgttgc tgaagatctc ttgcagatag cagatccggt tcttccgtct
     3841 ggtgtatctt cttctggcgg ttctcttcag ccgggtggcc tcggctgttt cgccgctgtc
     3901 gaacagcagg gctccgatca ggttcttctt gatgctgtgc cggtcggtgt tgcccagcac
     3961 cttgaatttc ttgctgggca ccttgtactc gtcggtgatc acggcccagc ccacagagtt
     4021 ggtgccgatg tccaggccga tgctgtactt cttgtcggct gctgggactc cgtggatacc
     4081 gaccttccgc ttcttctttg gggccatctt atcgtcatcg tctttgtaat caatatcatg
     4141 atccttgtag tctccgtcgt ggtccttata gtccatggtg gcaccggtcc aacctgaaaa
     4201 aaagtgattt caggcaggtg ctccaggtaa ttaaacatta ataccccacc aaccaaccat
     4261 cccttaaacc cttacctctt gctcagctaa ttacagcccg gaggagaagg gccgtcccgc
     4321 ccgctcacct gtgggagtaa cgcggtcagt cagagccggg gcgggcggcg cgaggcggcg
     4381 gcggagcggg gcacggggcg aaggcagcgc gcagcgactc ccgcccgccg cgcgcttcgc
     4441 tttttatagg gccgccgccg ccgccgcctc gccataaaag gaaactttcg gagcgcgccg
     4501 ctctgattgg ctgccgccgc acctctccgc ctcgccccgc cccgcccctc gccccgcccc
     4561 gccccccccc cccccccccc cccccccccc cccccccccc cccccccccc ccaaaaataa
     4621 aaaaaaaaaa aaaaaaaaca tagggggggg tggggagggg ggggagatgg ggagagtgaa
     4681 gcagaacgtg gggctcacct cgaccatggt aatagcgatg actaatacgt agatgtactg
     4741 ccaagtagga aagtcccata aggtcatgta ctgggcacaa tgccaggcgg gccatttacc
     4801 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg
     4861 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
     4921 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     4981 ttatgtaacg ggtacctcta gagccatttg tctgcagaat tggcgcacgc gctaaaaacg
     5041 gactagcctt attttaactt gctatttcta gctctaaaac aaaaaagcac cgactcggtg
     5101 ccactttttc aagttgataa cggactagcc ttatttcaac ttgctatttc tagctctgaa
     5161 acaggtcttc tcgaagaccc ggtgtttcgt cctttccaca agatatataa agccaagaaa
     5221 tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca taattttaaa
     5281 actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac taatatcttt
     5341 gtgtttacag tcaaattaat tccaattatc tctctaacag ccttgtatcg tatatgcaaa
     5401 tatgaaggaa tcatgggaaa taggccctca catgtgagca aaaggccagc aaaaggccag
     5461 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5521 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5581 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5641 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5701 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5761 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5821 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5881 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5941 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6001 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6061 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6121 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6181 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6241 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6301 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6361 atctggcccc agtgctgcaa tgataccgcg gcttccacgc tcaccggctc cagatttatc
     6421 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6481 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6541 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6601 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6661 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6721 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6781 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6841 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6901 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6961 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7021 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7081 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7141 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7201 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     7261 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     7321 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     7381 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     7441 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accataaaat
     7501 tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     7561 taaccaatag accgaaatcg gcaaaatccc ttataaatca aaagaatagc ccgagataga
     7621 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     7681 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat cacccaaatc
     7741 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     7801 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     7861 aggagcgggc gctaaggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     7921 cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg tatgcggtgt
     7981 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgcccctg caggcagctg
     8041 cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt
     8101 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
     8161 ggttcctgcg gccgctcccc agcatgcctg ctattctctt cccaatcctc ccccttgctg
     8221 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
     8281 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
     8341 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcga
     8401 gctctagtta gaattccttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     8461 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
     8521 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     8581 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt
     8641 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt
     8701 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     8761 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
     8821 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     8881 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca
     8941 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca
     9001 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     9061 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     9121 ccttgctcac tgggccagga ttctcctcga cgtcaccgca tgttagcaga cttcctctgc
     9181 cctctccact gccgaattcc tttttctttt ttgcctggcc ggcctttttc gtggccgccg
     9241 gccttttgtc gcctcccagc tgagacaggt cgatccgtgt ctcgtacagg cc
//
