LOCUS       sequence-261338-       12359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  H1 promoter expresses customizable Saur-guide; EF1a promoter 
            expresses SpyoCas9 and 2A site provides blasticidin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12359)
  AUTHORS   DeWeirdt PC, Sangree AK, Hanna RE, Sanson KR, Hegde M, Strand C, 
            Persky NS, Doench JG
  TITLE     Genetic screens in isogenic mammalian cell lines without single cell
            cloning.
  JOURNAL   Nat Commun. 2020 Feb 6;11(1):752. doi: 10.1038/s41467-020-14620-6.
  PUBMED    32029722
REFERENCE   2  (bases 1 to 12359)
  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: "2020-02-6"; volume: "11(1):752. doi"; pages: " 
            10.1038/s41467-020-14620-6"
COMMENT     Sequence Label: pXPR_213
FEATURES             Location/Qualifiers
     source          1..12359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    47..172
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    665..898
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1083..1127
                     /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"
     promoter        1433..1527
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     1444..1463
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    1481..1499
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1509..1527
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        1553..1628
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     misc_feature    1676..1793
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1932..3110
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2163..3101
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3058..3078
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3127..3147
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3154..7254
                     /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             3154..7254
                     /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             7261..7308
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7321..7377
                     /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             7387..7785
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7804..8392
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7857..7877)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8275..8286)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8464..8697
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8775..8896
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8812..8831)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8866..8885
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8936..9071
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9091..9110)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9092..9110)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9120..9137)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9120..9136)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9129..9151)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9278..9733
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9365..9384)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9575..9596
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9759..9863
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9864..9932,9933..10724)
                     /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(10082..10101)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10895..11483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11384..11403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11637..11654
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11771..11792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11807..11812,11813..11830,11831..11837)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11845..11861
                     /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     11850..11872
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11869..11885
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11869..11885
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11904..11924
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11906..11924
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        11952..12178
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             12179..12359
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
       61 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
      121 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
      181 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
      241 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
      301 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
      361 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
      421 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
      481 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
      541 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
      601 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
      661 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
      721 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
      781 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
      841 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
      901 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
      961 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     1021 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     1081 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     1141 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     1201 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     1261 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cggtacccga acgctgacgt
     1321 catcaacccg ctccaaggaa tcgcgggccc agtgtcacta ggcgggaaca cccagcgcgc
     1381 gtgcgccctg gcaggaagat ggctgtgagg gacaggggag tggcgccctg caatatttgc
     1441 atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tccctatcag tgatagagac
     1501 ttataagttc cctatcagtg atagagacac cggagacgct tttttcgtct cagtttaagt
     1561 actctggaaa cagaatctac ttaaacaagg caaaatgccg tgtttatctc gtcaacttgt
     1621 tggcgagatt ttttgaattc tagatcttga gacaaatggc agtattcatc cacaatttta
     1681 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
     1741 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
     1801 attacaggga cagcagagat ccactttggc gccggctcga gggggcccgg gtgcaaagat
     1861 ggataaagtt ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag
     1921 gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     1981 agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa
     2041 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     2101 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     2161 aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg
     2221 tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg
     2281 gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag
     2341 ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct
     2401 gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc
     2461 tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg
     2521 tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc
     2581 ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg
     2641 ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc
     2701 ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct
     2761 caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa
     2821 gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca
     2881 ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt
     2941 tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc
     3001 acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca
     3061 agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga gggatccacc
     3121 atggcaccaa agaagaagcg gaaggtcgca gccgacaaga agtacagcat cggcctggac
     3181 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     3241 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     3301 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     3361 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     3421 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     3481 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     3541 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     3601 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     3661 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     3721 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtaga cgccaaggcc
     3781 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     3841 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     3901 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     3961 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     4021 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     4081 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     4141 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     4201 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     4261 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     4321 gtgaagctga acagagagga cctgctgcgg aagcagcgta cgttcgacaa cggcagcatc
     4381 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     4441 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     4501 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     4561 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgctag cgcccagagc
     4621 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     4681 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     4741 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     4801 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     4861 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     4921 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     4981 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     5041 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     5101 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     5161 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     5221 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     5281 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     5341 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     5401 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     5461 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     5521 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     5581 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     5641 aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac
     5701 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     5761 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     5821 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     5881 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     5941 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     6001 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     6061 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     6121 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     6181 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     6241 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     6301 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     6361 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     6421 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     6481 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     6541 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     6601 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     6661 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     6721 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     6781 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     6841 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     6901 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     6961 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     7021 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     7081 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     7141 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag
     7201 agcatcaccg gcctgtacga gacacgaatc gatttgtctc agctgggagg cgacgccgca
     7261 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggc agggtccggg
     7321 gccaccaact tttccctgct gaagcaggct ggcgacgtcg aggagaaccc tggccctggc
     7381 tccggaatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct
     7441 acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc
     7501 gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa
     7561 ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg
     7621 atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc
     7681 gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt
     7741 gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctgatagac gcgttaagtc
     7801 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     7861 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     7921 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     7981 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     8041 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     8101 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     8161 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     8221 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     8281 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     8341 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     8401 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     8461 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     8521 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     8581 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     8641 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     8701 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     8761 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     8821 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     8881 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     8941 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     9001 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     9061 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     9121 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     9181 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     9241 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     9301 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     9361 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     9421 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     9481 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     9541 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     9601 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     9661 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     9721 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     9781 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     9841 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     9901 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     9961 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
    10021 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
    10081 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
    10141 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
    10201 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
    10261 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
    10321 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
    10381 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
    10441 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
    10501 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
    10561 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
    10621 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
    10681 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
    10741 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
    10801 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
    10861 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
    10921 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
    10981 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
    11041 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
    11101 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
    11161 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
    11221 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
    11281 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
    11341 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
    11401 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
    11461 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
    11521 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
    11581 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
    11641 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
    11701 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
    11761 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
    11821 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
    11881 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
    11941 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
    12001 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag
    12061 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac
    12121 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg
    12181 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
    12241 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
    12301 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagca
//
