LOCUS       sequence-352522-       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_152
FEATURES             Location/Qualifiers
     source          1..12797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..62
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     296..313
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    430..451
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(466..471,472..489,490..496)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    504..520
                     /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     509..531
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     528..544
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     528..544
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     563..583
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        565..583
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        611..837
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             838..1018
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1065..1190
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1683..1916
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2101..2145
                     /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    2379..2496
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2635..3813
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2866..3804
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3761..3781
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3830..3850
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3857..6394,6395..6397,6398..6859,6860..6862,6863..7030,
                     7031..7033,7034..7957)
                     /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             7964..8011
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8024..8077
                     /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             8084..8479
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             8480..8536
                     /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             8543..9241
                     /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    9260..9848
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9313..9333)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9731..9742)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9920..10153
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    10231..10352
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10268..10287)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10322..10341
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10392..10527
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10547..10566)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10548..10566)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10576..10593)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10576..10592)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10585..10607)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10734..11189
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10821..10840)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11031..11052
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11215..11319
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11320..11388,11389..12180)
                     /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(11538..11557)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(12351..12797,1..142)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
       61 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      121 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      181 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      241 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      301 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
      361 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      421 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
      481 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
      541 tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct
      601 gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt
      661 tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg
      721 tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc
      781 actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg
      841 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      901 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      961 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     1021 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
     1081 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
     1141 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
     1201 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1261 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1321 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1381 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1441 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1501 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1561 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1621 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1681 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1741 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1801 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1861 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1921 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1981 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     2041 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     2101 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     2161 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     2221 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2281 ttatcgtttc agacccacct cccaaccccg aggggaccct ggtacccatg catgcatgaa
     2341 ttctagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     2401 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     2461 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     2521 gatccacttt ggcgccggct cgagggggcc cgggtgcaaa gatggataaa gttttaaaca
     2581 gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc
     2641 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     2701 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     2761 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     2821 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt
     2881 ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc
     2941 acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt
     3001 tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg
     3061 cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat
     3121 aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat
     3181 agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc
     3241 ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg
     3301 ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc
     3361 gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg
     3421 tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg
     3481 cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca
     3541 gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc
     3601 tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt
     3661 ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc
     3721 ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt
     3781 caaagttttt ttcttccatt tcaggtgtcg tgagggatcc accatggcac caaagaagaa
     3841 gcggaaggtc gcagccgaca agaagtacag catcggcctg gacatcggca ccaactctgt
     3901 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
     3961 caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg
     4021 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
     4081 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
     4141 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
     4201 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
     4261 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
     4321 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
     4381 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
     4441 cgaggaaaac cccatcaacg ccagcggcgt agacgccaag gccatcctgt ctgccagact
     4501 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
     4561 cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
     4621 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
     4681 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
     4741 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
     4801 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
     4861 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
     4921 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat
     4981 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
     5041 ggacctgctg cggaagcagc gtacgttcga caacggcagc atcccccacc agatccacct
     5101 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
     5161 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
     5221 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
     5281 gaacttcgag gaagtggtgg acaagggcgc tagcgcccag agcttcatcg agcggatgac
     5341 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
     5401 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
     5461 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa
     5521 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
     5581 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
     5641 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
     5701 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
     5761 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
     5821 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
     5881 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
     5941 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
     6001 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
     6061 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
     6121 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
     6181 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
     6241 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
     6301 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
     6361 cgatgtggac catatcgtgc ctcagagctt tctggccgac gactccatcg acaacaaggt
     6421 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
     6481 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa
     6541 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
     6601 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
     6661 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat
     6721 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
     6781 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
     6841 cctgatcaaa aagtaccctg ccctggaaag cgagttcgtg tacggcgact acaaggtgta
     6901 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
     6961 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
     7021 gatccggaag gcccctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa
     7081 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa
     7141 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
     7201 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
     7261 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
     7321 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
     7381 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
     7441 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
     7501 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
     7561 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
     7621 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt
     7681 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa
     7741 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
     7801 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
     7861 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
     7921 cgagacacga atcgatttgt ctcagctggg aggcgacgcc gcaaaaaggc cggcggccac
     7981 gaaaaaggcc ggccaggcaa aaaagaaaaa ggcaggctca ggcgagggca gaggaagtct
     8041 gctaacatgc ggtgacgtcg aggagaatcc tggcccaagc gctatggcca agcctttgtc
     8101 tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc
     8161 tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt
     8221 caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc
     8281 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat
     8341 cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc
     8401 catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc
     8461 tggttatgtg tgggagggcg caacaaactt ctctctgctg aaacaagccg gagatgtcga
     8521 agagaatcct ggaccgacta gtatggtgag cgagctgatt aaggagaaca tgcacatgaa
     8581 gctatacatg gagggcaccg tgaacaacca ccacttcaag tgcacatccg agggcgaagg
     8641 caagccctac gagggcaccc agaccatgag aatcaaggcg gtcgagggcg gccctctccc
     8701 cttcgccttc gacatcctgg ctaccagctt catgtacggc agcaaaacct tcatcaacca
     8761 cacccagggc atccccgact tctttaagca gtccttcccc gagggcttca catgggagag
     8821 agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga
     8881 cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcccatcca acggccctgt
     8941 gatgcagaag aaaacactcg gctgggaggc ctccaccgag accctgtacc ccgctgacgg
     9001 cggcctggaa ggcagagccg acatggccct gaagctcgtg ggcgggggcc acctgatctg
     9061 caacttgaag accacataca gatccaagaa acccgctaag aacctcaaga tgcccggcgt
     9121 ctactatgtg gacagaagac tggaaagaat caaggaggcc gacaaagaga cctacgtcga
     9181 gcagcacgag gtggctgtgg ccagatactg cgacctccct agcaaactgg ggcacagatg
     9241 atagacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     9301 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     9361 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     9421 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     9481 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     9541 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     9601 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     9661 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     9721 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     9781 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     9841 ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     9901 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     9961 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
    10021 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
    10081 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
    10141 aaaatctcta gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt
    10201 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
    10261 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
    10321 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
    10381 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
    10441 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
    10501 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
    10561 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
    10621 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
    10681 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
    10741 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
    10801 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
    10861 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
    10921 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
    10981 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
    11041 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
    11101 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
    11161 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
    11221 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
    11281 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
    11341 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
    11401 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
    11461 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
    11521 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
    11581 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
    11641 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
    11701 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
    11761 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
    11821 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
    11881 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
    11941 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
    12001 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
    12061 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
    12121 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
    12181 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
    12241 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
    12301 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
    12361 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
    12421 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
    12481 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
    12541 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
    12601 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
    12661 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
    12721 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
    12781 gacaggtatc cggtaag
//
