LOCUS       sequence-352531-       12797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  nuclease Cas expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12797)
  AUTHORS   Sangree AK, Griffith AL, Szegletes ZM, Roy P, DeWeirdt PC, Hegde M, 
            McGee AV, Hanna RE, Doench JG
  TITLE     Benchmarking of SpCas9 variants enables deeper base editor screens 
            of BRCA1 and BCL2.
  JOURNAL   Nat Commun. 2022 Mar 14;13(1):1318. doi: 10.1038/s41467-022-28884-7.
  PUBMED    35288574
REFERENCE   2  (bases 1 to 12797)
  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: "Nat 
            Commun."; date: "2022-03-14"; volume: "13(1):1318. doi"; pages: " 
            10.1038/s41467-022-28884-7"
COMMENT     Sequence Label: pRDA_450
FEATURES             Location/Qualifiers
     source          1..12797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    88..109
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(124..129,130..147,148..154)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    162..178
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     167..189
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     186..202
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     186..202
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     221..241
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        223..241
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        269..495
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             496..676
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    723..848
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1341..1574
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1759..1803
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2037..2154
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2293..3471
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2524..3462
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3419..3439
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3488..3508
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3515..7615
                     /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
                     LIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7622..7669
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7682..7735
                     /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             7742..8137
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             8138..8194
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             8201..8899
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     misc_feature    8918..9506
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8971..8991)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9389..9400)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9578..9811
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    9889..10010
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9926..9945)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9980..9999
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10050..10185
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10205..10224)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10206..10224)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10234..10251)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10234..10250)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10243..10265)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10392..10847
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10479..10498)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10689..10710
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10873..10977
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10978..11046,11047..11838)
                     /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(11196..11215)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12009..12597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12498..12517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12751..12768
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
       61 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      121 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      181 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
      241 ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc
      301 aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc
      361 cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg
      421 acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta
      481 gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc
      541 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
      601 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
      661 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
      721 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
      781 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
      841 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
      901 cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac
      961 gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac
     1021 agctacaacc atcccttcag acaggatcag aagaacttag atcattatat aatacagtag
     1081 caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca
     1141 agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc
     1201 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     1261 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     1321 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     1381 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     1441 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     1501 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     1561 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     1621 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     1681 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     1741 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     1801 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     1861 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     1921 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggaccctgg
     1981 tacccatgca tgcatgaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt
     2041 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     2101 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     2161 tattacaggg acagcagaga tccactttgg cgccggctcg agggggcccg ggtgcaaaga
     2221 tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa
     2281 ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     2341 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
     2401 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     2461 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     2521 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc
     2581 gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt
     2641 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga
     2701 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
     2761 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
     2821 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg
     2881 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
     2941 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
     3001 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
     3061 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc
     3121 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     3181 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     3241 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg
     3301 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg
     3361 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc
     3421 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg agggatccac
     3481 catggcacca aagaagaagc ggaaggtcgc agccgacaag aagtacagca tcggcctgga
     3541 catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa
     3601 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc
     3661 cctgctgttc gacagcggcg aaacagccga gagaacccgg ctgaagagaa ccgccagaag
     3721 aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat
     3781 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
     3841 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga
     3901 gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga
     3961 cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat
     4021 cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca
     4081 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtag acgccaaggc
     4141 catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc
     4201 cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc
     4261 caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac
     4321 ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt
     4381 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
     4441 cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca
     4501 ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat
     4561 tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga
     4621 agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct
     4681 cgtgaagctg aacagagagg acctgctgcg gaagcagcgt acgttcgaca acggcagcat
     4741 cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta
     4801 cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta
     4861 ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga
     4921 ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgcta gcgcccagag
     4981 cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa
     5041 gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt
     5101 gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga
     5161 cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa
     5221 gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc
     5281 cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga
     5341 ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga
     5401 gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca
     5461 gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat
     5521 ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
     5581 cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa
     5641 agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag
     5701 ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt
     5761 gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
     5821 ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
     5881 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
     5941 gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat
     6001 caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga
     6061 ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt
     6121 gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa
     6181 gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga
     6241 actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca
     6301 cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat
     6361 ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt
     6421 ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa
     6481 cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta
     6541 cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg
     6601 caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat
     6661 taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg
     6721 ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc
     6781 ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat
     6841 cagacccaag aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa
     6901 gtacggcggc ttcctgtggc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga
     6961 aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga
     7021 aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt
     7081 gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg
     7141 gaagagaatg ctggcctctg ccaagcagct gcagaaggga aacgaactgg ccctgccctc
     7201 caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga
     7261 ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat
     7321 cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt
     7381 gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat
     7441 ccacctgttt accctgacca gactgggagc ccctagagcc ttcaagtact ttgacaccac
     7501 catcgacccc aagcagtaca gaagcaccaa agaggtgctg gacgccaccc tgatccacca
     7561 gagcatcacc ggcctgtacg agacacgaat cgatttgtct cagctgggag gcgacgccgc
     7621 aaaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg caggctcagg
     7681 cgagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaagcgc
     7741 tatggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat
     7801 caacagcatc cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg
     7861 ccgcatcttc actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt
     7921 ggtgctgggc actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg
     7981 aaatgagaac aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct
     8041 gcatcctggg atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat
     8101 tcgtgaattg ctgccctctg gttatgtgtg ggagggcgca acaaacttct ctctgctgaa
     8161 acaagccgga gatgtcgaag agaatcctgg accgactagt atggtgagcg agctgattaa
     8221 ggagaacatg cacatgaagc tatacatgga gggcaccgtg aacaaccacc acttcaagtg
     8281 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggcggt
     8341 cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag
     8401 caaaaccttc atcaaccaca cccagggcat ccccgacttc tttaagcagt ccttccccga
     8461 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
     8521 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
     8581 cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct ccaccgagac
     8641 cctgtacccc gctgacggcg gcctggaagg cagagccgac atggccctga agctcgtggg
     8701 cgggggccac ctgatctgca acttgaagac cacatacaga tccaagaaac ccgctaagaa
     8761 cctcaagatg cccggcgtct actatgtgga cagaagactg gaaagaatca aggaggccga
     8821 caaagagacc tacgtcgagc agcacgaggt ggctgtggcc agatactgcg acctccctag
     8881 caaactgggg cacagatgat agacgcgtta agtcgacaat caacctctgg attacaaaat
     8941 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     9001 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     9061 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     9121 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     9181 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     9241 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     9301 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     9361 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     9421 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     9481 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     9541 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     9601 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     9661 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     9721 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     9781 cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt
     9841 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     9901 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     9961 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
    10021 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
    10081 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
    10141 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
    10201 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
    10261 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
    10321 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
    10381 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
    10441 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
    10501 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
    10561 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
    10621 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
    10681 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
    10741 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
    10801 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
    10861 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
    10921 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
    10981 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
    11041 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
    11101 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
    11161 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
    11221 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
    11281 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
    11341 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
    11401 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
    11461 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
    11521 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
    11581 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
    11641 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
    11701 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
    11761 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
    11821 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
    11881 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
    11941 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
    12001 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
    12061 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
    12121 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
    12181 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
    12241 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
    12301 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
    12361 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
    12421 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
    12481 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
    12541 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
    12601 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
    12661 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
    12721 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
    12781 cgcccaatac gcaaacc
//
