LOCUS       sequence-256463-       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"
     promoter        87..1265
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          318..1256
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1213..1233
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1282..1302
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1309..5409
                     /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             1309..5409
                     /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             5416..5463
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5476..5532
                     /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             5542..5940
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5959..6547
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6012..6032)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6430..6441)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6619..6852
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6930..7051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6967..6986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7021..7040
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7091..7226
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7246..7265)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7247..7265)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7275..7292)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7275..7291)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7284..7306)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7433..7888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7520..7539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7730..7751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7914..8018
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8019..8087,8088..8879)
                     /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(8237..8256)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9050..9638
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9539..9558
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9792..9809
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9926..9947
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9962..9967,9968..9985,9986..9992)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10000..10016
                     /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     10005..10027
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10024..10040
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10024..10040
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10059..10079
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10061..10079
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        10107..10333
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             10334..10514
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    10561..10686
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11179..11412
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             11597..11641
                     /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        11947..12041
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     11958..11977
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    11995..12013
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12023..12041
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        12067..12142
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     misc_feature    12190..12307
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct
       61 tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat
      121 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
      181 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
      241 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
      301 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg
      361 gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc
      421 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt
      481 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt
      541 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat
      601 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc
      661 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca
      721 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
      781 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg
      841 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc
      901 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac
      961 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt
     1021 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag
     1081 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag
     1141 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat
     1201 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt
     1261 cgtgagggat ccaccatggc accaaagaag aagcggaagg tcgcagccga caagaagtac
     1321 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     1381 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     1441 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     1501 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     1561 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     1621 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     1681 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     1741 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     1801 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     1861 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     1921 gtagacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     1981 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     2041 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     2101 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     2161 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     2221 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     2281 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     2341 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     2401 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     2461 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcgtacgttc
     2521 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     2581 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     2641 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     2701 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     2761 gctagcgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     2821 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     2881 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     2941 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     3001 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     3061 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     3121 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     3181 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     3241 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     3301 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     3361 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     3421 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     3481 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     3541 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     3601 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     3661 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     3721 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     3781 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     3841 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     3901 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     3961 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     4021 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     4081 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     4141 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     4201 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     4261 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     4321 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     4381 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     4441 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     4501 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     4561 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     4621 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     4681 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     4741 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     4801 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     4861 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     4921 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     4981 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     5041 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     5101 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     5161 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     5221 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     5281 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     5341 accctgatcc accagagcat caccggcctg tacgagacac gaatcgattt gtctcagctg
     5401 ggaggcgacg ccgcaaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa
     5461 aaggcagggt ccggggccac caacttttcc ctgctgaagc aggctggcga cgtcgaggag
     5521 aaccctggcc ctggctccgg aatggccaag cctttgtctc aagaagaatc caccctcatt
     5581 gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc
     5641 gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg
     5701 ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg
     5761 acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgc
     5821 cgacaggtgc ttctcgatct gcatcctggg atcaaagcca tagtgaagga cagtgatgga
     5881 cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctga
     5941 tagacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     6001 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     6061 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     6121 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     6181 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     6241 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     6301 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     6361 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     6421 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     6481 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     6541 tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     6601 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt
     6661 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     6721 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     6781 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     6841 aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg
     6901 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     6961 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     7021 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     7081 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     7141 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     7201 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     7261 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     7321 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     7381 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     7441 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     7501 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     7561 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     7621 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     7681 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     7741 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     7801 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     7861 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     7921 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     7981 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     8041 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     8101 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     8161 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     8221 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     8281 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     8341 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     8401 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     8461 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     8521 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     8581 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     8641 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     8701 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     8761 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     8821 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     8881 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     8941 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     9001 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     9061 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     9121 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     9181 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     9241 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     9301 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     9361 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     9421 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     9481 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     9541 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     9601 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     9661 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     9721 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     9781 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     9841 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     9901 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     9961 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
    10021 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
    10081 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa
    10141 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg
    10201 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac
    10261 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc
    10321 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
    10381 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
    10441 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
    10501 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
    10561 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
    10621 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
    10681 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
    10741 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
    10801 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
    10861 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
    10921 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
    10981 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
    11041 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
    11101 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
    11161 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
    11221 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
    11281 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
    11341 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
    11401 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
    11461 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
    11521 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
    11581 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
    11641 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
    11701 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
    11761 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccggtac
    11821 ccgaacgctg acgtcatcaa cccgctccaa ggaatcgcgg gcccagtgtc actaggcggg
    11881 aacacccagc gcgcgtgcgc cctggcagga agatggctgt gagggacagg ggagtggcgc
    11941 cctgcaatat ttgcatgtcg ctatgtgttc tgggaaatca ccataaacgt gaaatcccta
    12001 tcagtgatag agacttataa gttccctatc agtgatagag acaccggaga cgcttttttc
    12061 gtctcagttt aagtactctg gaaacagaat ctacttaaac aaggcaaaat gccgtgttta
    12121 tctcgtcaac ttgttggcga gattttttga attctagatc ttgagacaaa tggcagtatt
    12181 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
    12241 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
    12301 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgaggggg
//
