LOCUS       Exported               11040 bp ds-DNA     circular SYN 21-MAR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pX461-evoCDA1-SpCas9-BpiI mut-correct-Int-sgRNA1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11040)
  AUTHORS   NIH-NCI
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Mar 25, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..11040
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..19
                     /label=A192-px459v2-deltaSpCas9-FP
     primer_bind     1..19
                     /label=A219-px459v2-dSpCas9-XhoI-F
     CDS             16..69
                     /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             70..669
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(70..89)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     566..586
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    668..669
                     /label=Sanger sequencing confirmed silent mutation
     primer_bind     complement(697..714)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    703..910
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   919..1059
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   919..1048
                     /label=AAV2 ITR
     rep_origin      1134..1589
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1221..1240)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1431..1452
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1606..1625)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1725..1747
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1785..1803)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1845..1862)
                     /label=pQE promoter
     promoter        1871..1975
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1976..2836
                     /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(2194..2213)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3007..3595
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3228..3247
                     /label=RepO-R
     primer_bind     complement(3354..3372)
                     /label=RepO-F
     primer_bind     3496..3515
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3657..3897
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     3657..3677
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3701..3725
                     /label=TRC-F
     primer_bind     3828..3847
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    3906
                     /label=G added
                     /note="G added at the beginning of 5'end of gRNA"
     mRNA            3907..3926
                     /label=Intergenic sgRNA1
                     /label=guide-RNA
                     /note="/created_by=User"
     misc_RNA        3927..4002
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        4099..4384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        4386..4663
                     /label=chicken beta-actin promoter
     intron          4664..4891
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      4903..4912
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4912..4977
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     4965..5001
                     /label=Primer 3
     primer_bind     4965..5001
                     /label=A175-px461-evoCDA1-SpCas9-FP
     primer_bind     complement(4965..4983)
                     /label=A220-px459v2-dSpCas9-XhoI-R
     primer_bind     complement(4965..4983)
                     /label=A193-px459v2-deltaSpCas9-RP
     CDS             4965..4977
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DDDK"
     primer_bind     4981..5000
                     /label=EDb 1.4-CBE_F
     primer_bind     4981..5000
                     /label=EDb 4.4L-CBE_F
     misc_feature    4984..5604
                     /label=evoCDA1
     primer_bind     5095..5141
                     /label=Fragment 1.FOR
     primer_bind     5498..5519
                     /label=Fragment 2.FOR
     primer_bind     complement(5498..5517)
                     /label=Fragment 1.REV
     CDS             5515..5517
                     /codon_start=1
                     /label=lys codon mutated
                     /translation="K"
     primer_bind     complement(5591..5614)
                     /label=EDb 1.4-CBE_R
     primer_bind     complement(5591..5614)
                     /label=EDb 4.4L-CBE_R
     primer_bind     5595..5623
                     /label=EDb 1.4-DBD_F
     primer_bind     5595..5623
                     /label=EDb 4.4L-DBD_F
     primer_bind     5598..5635
                     /label=EDb 4.0L (not 4.-1L)-DBD_F
     primer_bind     5598..5629
                     /label=EDb 1.0 (not 1.-1)-DBD_F
     CDS             5604
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation=""
     misc_feature    5605..5700
                     /label=Linker
     primer_bind     5605..5623
                     /label=EDb 1.3-DBD_F
     primer_bind     5605..5623
                     /label=EDb 4.3L-DBD_F
     misc_feature    5701..6042
                     /label=ss DNA-binding domain
     primer_bind     5785..5805
                     /label=ssDBD seq F
     CDS             6043..6138
                     /codon_start=1
                     /label=Linker
                     /translation="SGGSSGGSSGSETPGTSESATPESSGGSSGGS"
     primer_bind     complement(6111..6156)
                     /label=EDb 1.0 (not 1.-1)-DBD_R
     primer_bind     complement(6114..6132)
                     /label=EDb 4.0L (not 4.-1L)-DBD_R
     primer_bind     complement(6118..6156)
                     /label=EDb 1.1_R
     primer_bind     complement(6118..6156)
                     /label=EDb 1.2_R
     primer_bind     complement(6118..6144)
                     /label=EDb 4.3L-DBD_R
     primer_bind     complement(6118..6144)
                     /label=EDb 4.4L-DBD_R
     primer_bind     complement(6118..6144)
                     /label=EDb 4.1L_R
     primer_bind     complement(6120..6166)
                     /label=Fragment 2.REV
     primer_bind     complement(6122..6161)
                     /label=EDb 1.3-DBD_R
     primer_bind     complement(6122..6161)
                     /label=EDb 1.4-DBD_R
     primer_bind     complement(6125..6144)
                     /label=EDb 4.2L_R
     primer_bind     6139..6166
                     /label=EDb 1.0/3.0_F
     CDS             6145..10245
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(6200..6220)
                     /label=Cas9 rev seq1
     primer_bind     6513..6532
                     /label=Cas9_confirm_2
     primer_bind     7113..7132
                     /label=Cas9_confirm_3
     primer_bind     7713..7732
                     /label=Cas9_confirm_4
     primer_bind     8293..8312
                     /label=Cas9_5_F
     primer_bind     8293..8312
                     /label=Cas9_5_F (1)
     primer_bind     8313..8328
                     /label=Cas9_confirm_5
     primer_bind     8496..8515
                     /label=Cas9 intern seq 3
     primer_bind     8913..8932
                     /label=Cas9_confirm_6
     primer_bind     9513..9535
                     /label=Cas9_confirm_7
     primer_bind     complement(9748..9768)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     10113..10137
                     /label=Cas9_comfirm_8
     primer_bind     complement(10233..10269)
                     /label=EDb 1.-1-Cas9_R
     primer_bind     complement(10241..10253)
                     /label=ED 3.0_R
     primer_bind     10250..10296
                     /label=EDb 1.-1/4.-1-UGI_F
     primer_bind     10276..10296
                     /label=EDb 1.0/4.0-UGI_F
     CDS             10282..10530
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             10561..10809
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     primer_bind     complement(10806..10845)
                     /label=Fragment.REV
     primer_bind     complement(10806..10843)
                     /label=EDb 1.0-UGI_R
     primer_bind     complement(10806..10821)
                     /label=Infusion fragment R
     primer_bind     10827..10851
                     /label=inf 6nls.FOR
     primer_bind     complement(10829..10845)
                     /label=Infusion vec R
     primer_bind     complement(10829..10843)
                     /label=ED 3.0_R
     CDS             10834..10860
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             10870..10896
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             10906..10932
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             10942..10968
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             10978..11004
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             11014..11040
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     primer_bind     complement(join(11018..11040,1..19))
                     /label=Primer 4
     primer_bind     complement(join(11018..11040,1..19))
                     /label=A179-px461-evoCDA1-SpCas9-RP
     primer_bind     complement(11020..11040)
                     /label=EDi 1-3_R
     primer_bind     complement(join(11022..11040,1))
                     /label=inf 6nls.REV
ORIGIN
        1 gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
       61 cctggcccaa tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
      121 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
      181 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
      241 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
      301 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
      361 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
      421 cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag
      481 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
      541 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
      601 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
      661 ggtgcctagg aattctaact agagctcgct gatcagcctc gactgtgcct tctagttgcc
      721 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
      781 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
      841 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc
      901 atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
      961 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     1021 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     1081 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     1141 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1201 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1261 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1321 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     1381 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1441 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     1501 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1561 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     1621 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     1681 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     1741 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     1801 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1861 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1921 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1981 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2041 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2101 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2161 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     2221 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2281 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2341 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2401 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2461 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2521 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2581 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2641 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     2701 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2761 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2821 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2881 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2941 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3001 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3061 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3121 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3181 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3241 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3301 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3361 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3421 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3481 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3541 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3601 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtgagg
     3661 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     3721 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     3781 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     3841 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     3901 acaccgttga ctgcacacaa ctgggcgttt tagagctaga aatagcaagt taaaataagg
     3961 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt tttagagcta
     4021 gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat tctgcagaca
     4081 aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     4141 caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca
     4201 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     4261 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta
     4321 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     4381 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
     4441 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
     4501 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag
     4561 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
     4621 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     4681 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     4741 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct
     4801 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct
     4861 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag
     4921 gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg
     4981 gccaccgacg ccgagtacgt gcggatccac gagaagctgg atatctatac attcaagaag
     5041 cagtttagca acaataagaa gtccgtgtct cacagatgct acgtgctgtt cgagctgaag
     5101 cggagaggag agaggcgcgc ctgtttttgg ggctatgccg tgaacaagcc acagtctgga
     5161 accgagaggg gaatccacgc agagatcttc agcatcagga aggtggagga gtacctgcgc
     5221 gacaaccccg gccagtttac aatcaattgg tatagctcct ggagcccttg cgccgattgt
     5281 gccgagaaga tcctggagtg gtacaaccag gagctgaggg gcaatggcca caccctgaag
     5341 atctgggtgt gcaagctgta ctatgagaag aacgccagga atcagatcgg cctgtggaac
     5401 ctgcgcgaca atggcgtggg cctgaacgtg atggtgtccg agcactatca gtgctgtcgc
     5461 aagatcttta tccagtctag ccacaatcag ctgaacgaga atcggtggct ggagaaaaca
     5521 ctgaagagag ccgagaagcg gagaagcgag ctgtccatca tgtttcaggt gaagatcctg
     5581 cacaccacaa agtctcccgc cgtgagcgga ggatctagcg gaggatcaag cggaagcgag
     5641 actcctggaa ccagcgaaag cgcaacccca gaaagcagcg gaggaagtag cggaggaagc
     5701 atggcaatgc agatgcagct tgaagcaaat gcagatactt cagtggaaga agaaagcttt
     5761 ggcccacaac ccatttcacg gttagagcag tgtggcataa atgccaacga tgtgaagaaa
     5821 ttggaagaag ctggattcca tactgtggag gctgttgcct atgcgccaaa gaaggagcta
     5881 ataaatatta agggaattag tgaagccaaa gctgataaaa ttctggctga ggcagctaaa
     5941 ttagttccaa tgggtttcac cactgcaact gaattccacc aaaggcggtc agagatcata
     6001 cagattacta ctggctccaa agagcttgac aaactacttc aatctggcgg atctagcggt
     6061 ggatctagcg gctctgagac ccctggaaca tccgaatccg ccactccaga gagcagcgga
     6121 ggctcttctg gaggatcaag cgctgacaag aagtacagca tcggcctggc catcggcacc
     6181 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     6241 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     6301 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     6361 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     6421 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     6481 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     6541 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     6601 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     6661 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     6721 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     6781 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     6841 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     6901 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     6961 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     7021 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     7081 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     7141 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     7201 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     7261 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     7321 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     7381 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     7441 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     7501 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     7561 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     7621 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     7681 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     7741 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     7801 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     7861 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     7921 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     7981 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     8041 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     8101 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     8161 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     8221 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     8281 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     8341 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     8401 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     8461 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     8521 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     8581 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     8641 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     8701 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     8761 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     8821 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     8881 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     8941 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     9001 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     9061 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     9121 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     9181 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     9241 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     9301 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     9361 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     9421 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     9481 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     9541 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     9601 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     9661 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     9721 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     9781 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     9841 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     9901 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     9961 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
    10021 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
    10081 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
    10141 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
    10201 ggcctgtacg agacacggat cgacctgtct cagctgggag gtgacagtgg cggaagtgga
    10261 ctagtaggaa gtggcgggag cactaatctg agcgacatca ttgagaagga gactgggaaa
    10321 cagctggtca ttcaggagtc catcctgatg ctgcctgagg aggtggagga agtgatcggc
    10381 aacaagccag agtctgacat cctggtgcac accgcctacg acgagtccac agatgagaat
    10441 gtgatgctgc tgacctctga cgcccccgag tataagcctt gggccctggt catccaggat
    10501 tctaacggcg agaataagat caagatgctg agcggaggat ccggaggatc tggaggcagc
    10561 accaacctgt ctgacatcat cgagaaggag acaggcaagc agctggtcat ccaggagagc
    10621 atcctgatgc tgcccgaaga agtcgaagaa gtgatcggaa acaagcctga gagcgatatc
    10681 ctggtccata ccgcctacga cgagagtacc gacgaaaatg tgatgctgct gacatccgac
    10741 gccccagagt ataagccctg ggctctggtc atccaggatt ccaacggaga gaacaaaatc
    10801 aaaatgctgt ctggcggctc agaattcgga tcccctgctg ctaaacgtgt taagcttgat
    10861 gggggtagcc cggcagccaa gagagtcaaa ctcgacggcg gctccccagc ggcaaaaagg
    10921 gtgaaactag acgggggtag ccccgccgcg aagcgtgtaa agctggatgg aggatcgcct
    10981 gcggctaagc gagtcaaatt agacggtggg tcaccggctg caaagcgcgt caaattggat
//
