LOCUS       sequence-413997-       12209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains Cas9 gRNA cloning site, inducible Cas9, rtTA-T2A-puro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12209)
  AUTHORS   Nuttle X, Burt ND, Currall B, Moyses-Oliveira M, Mohajeri K, Bhavsar
            R, Lucente D, Yadav R, Tai DJC, Gusella JF, Talkowski ME
  TITLE     Parallelized engineering of mutational models using piggyBac 
            transposon delivery of CRISPR libraries.
  JOURNAL   Cell Rep Methods. 2023 Dec 5:100672. doi: 
            10.1016/j.crmeth.2023.100672.
  PUBMED    38091988
REFERENCE   2  (bases 1 to 12209)
  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: "Cell Rep 
            Methods. 2023 Dec 5:100672. doi: 10.1016/j.crmeth.2023.100672."
COMMENT     Sequence Label: pPB9v1
FEATURES             Location/Qualifiers
     source          1..12209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..67
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        554..802
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     554..574
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     725..744
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        821..896
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        960..1338
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    968..986
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1004..1022
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1040..1058
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1076..1094
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1112..1130
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1148..1166
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1184..1202
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1247..1271
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1365..1374
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1377..1397
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1422..5522
                     /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             1422..5522
                     /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             5523..5570
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5625..5644)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5669..5717
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        5948..6159
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             6172..6440
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             6474..7217
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             7218..7271
                     /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             7272..7871
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7272..7291)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7768..7788
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7904..8025
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7941..7960)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7995..8014
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   8794..8828
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9319..9335)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9319..9335)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9332..9354)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9343..9359
                     /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)."
     promoter        complement(join(9367..9373,9374..9391,9392..9397))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9412..9433
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9550..9567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9721..10309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9801..9820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10480..11271,11272..11340))
                     /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     11103..11122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11341..11445)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11471..11926)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11608..11629)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     11820..11839
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12053..12075
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12067..12084
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12068..12084
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg tgtaaaattg
       61 acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa
      121 gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt
      181 tatttattta ttaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt
      241 atgagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
      301 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
      361 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
      421 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaggcctc
      481 cacggccaga ctagaggtac caacgcggcc tttttacggt tcctggcctt ttgctggcct
      541 tttgctcaca tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      601 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      661 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      721 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      781 cttgtggaaa ggacgaaaca ccgggtcttc gagaagacct gttttagagc tagaaatagc
      841 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      901 ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagctcg
      961 agtttactcc ctatcagtga tagagaacgt atgaagagtt tactccctat cagtgataga
     1021 gaacgtatgc agactttact ccctatcagt gatagagaac gtataaggag tttactccct
     1081 atcagtgata gagaacgtat gaccagttta ctccctatca gtgatagaga acgtatctac
     1141 agtttactcc ctatcagtga tagagaacgt atatccagtt tactccctat cagtgataga
     1201 gaacgtataa gctttaggcg tgtacggtgg gcgcctataa aagcagagct cgtttagtga
     1261 accgtcagat cgcctggagc aattccacaa cacttttgtc ttataccaac tttccgtacc
     1321 acttcctacc ctcgtaaagg tctagagcta gcgaattcga atttgccacc atggccccaa
     1381 agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag tactccattg
     1441 ggctcgatat cggcacaaac agcgtcggct gggccgtcat tacggacgag tacaaggtgc
     1501 cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca cagcataaag aagaacctca
     1561 ttggcgccct cctgttcgac tccggggaga cggccgaagc cacgcggctc aaaagaacag
     1621 cacggcgcag atatacccgc agaaagaatc ggatctgcta cctgcaggag atcttcagca
     1681 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     1741 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     1801 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     1861 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     1921 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     1981 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     2041 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     2101 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     2161 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     2221 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     2281 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     2341 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     2401 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     2461 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     2521 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     2581 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     2641 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     2701 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     2761 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     2821 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     2881 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     2941 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     3001 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     3061 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     3121 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     3181 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     3241 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     3301 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     3361 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     3421 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     3481 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     3541 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     3601 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     3661 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     3721 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     3781 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     3841 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     3901 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     3961 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     4021 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     4081 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     4141 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     4201 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     4261 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     4321 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     4381 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     4441 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     4501 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     4561 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     4621 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     4681 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     4741 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     4801 ccaagaaata cggcggattc gattctccta cagtcgctta cagtgtactg gttgtggcca
     4861 aagtggagaa agggaagtct aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa
     4921 tcatggagcg atcaagcttc gaaaaaaacc ccatcgactt tctcgaggcg aaaggatata
     4981 aagaggtcaa aaaagacctc atcattaagc ttcccaagta ctctctcttt gagcttgaaa
     5041 acggccggaa acgaatgctc gctagtgcgg gcgagctgca gaaaggtaac gagctggcac
     5101 tgccctctaa atacgttaat ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt
     5161 ctcccgaaga taatgagcag aagcagctgt tcgtggaaca acacaaacac taccttgatg
     5221 agatcatcga gcaaataagc gaattctcca aaagagtgat cctcgccgac gctaacctcg
     5281 ataaggtgct ttctgcttac aataagcaca gggataagcc catcagggag caggcagaaa
     5341 acattatcca cttgtttact ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg
     5401 acaccaccat agacagaaag cggtacacct ctacaaagga ggtcctggac gccacactga
     5461 ttcatcagtc aattacgggg ctctatgaaa caagaatcga cctctctcag ctcggtggag
     5521 acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag tgaaagggtt
     5581 cgatccctac cggttagtaa tgagtttaaa cgggggaggc taactgaaac acggaaggag
     5641 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggg
     5701 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     5761 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     5821 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     5881 agcagatctg cgaaatcgga tccagctgaa tctaagtgga tctgcgatcg ctccggtgcc
     5941 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     6001 aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     6061 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     6121 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg aggggctcgc
     6181 atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg gttgagtcgc
     6241 gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta
     6301 aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc tagactcagc
     6361 cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt ttcgttttct
     6421 gttctgcgcc gttacagatc taagctgtga ccggcgccta ctctagagcc gccatgtcta
     6481 gactggacaa gagcaaagtc ataaactctg ctctggaatt actcaatgga gtcggtatcg
     6541 aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt tgagcagcct accctgtact
     6601 ggcacgtgaa gaacaagcgg gccctgctcg atgccctgcc aatcgagatg ctggacaggc
     6661 atcataccca ctcctgcccc ctggaaggcg agtcatggca agactttctg cggaacaacg
     6721 ccaagtcata ccgctgtgct ctcctctcac atcgcgacgg ggctaaagtg catctcggca
     6781 cccgcccaac agagaaacag tacgaaaccc tggaaaatca gctcgcgttc ctgtgtcagc
     6841 aaggcttctc cctggagaac gcactgtacg ctctgtccgc cgtgggccac tttacactgg
     6901 gctgcgtatt ggaggaacag gagcatcaag tagcaaaaga ggaaagagag acacctacca
     6961 ccgattctat gcccccactt ctgaaacaag caattgagct gttcgaccgg cagggagccg
     7021 aacctgcctt ccttttcggc ctggaactaa tcatatgtgg cctggagaaa cagctaaagt
     7081 gcgaaagcgg cgggccgacc gacgcccttg acgattttga cttagacatg ctcccagccg
     7141 atgcccttga cgactttgac cttgatatgc tgcctgctga cgctcttgac gattttgacc
     7201 ttgacatgct ccccggggag ggcagaggaa gtctactaac atgcggtgac gtggaggaga
     7261 atcccggccc tatgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     7321 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     7381 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     7441 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     7501 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     7561 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     7621 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
     7681 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     7741 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     7801 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     7861 ccggtgcctg aaatcaacct ctggattaca aaatttgtgg gtaaacttgt ttattgcagc
     7921 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     7981 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggaattg
     8041 actcaaatga tgtcaattag tctatcagaa gctcatctgg tctcccttcc gggggacaag
     8101 acatccctgt ttaatattta aacagcagtg ttcccaaact gggttcttat atcccttgct
     8161 ctggtcaacc aggttgcagg gtttcctgtc ctcacaggaa cgaagtccct aaagaaacag
     8221 tggcagccag gtttagcccc ggaattgact ggattccttt tttagggccg cggcccattg
     8281 gtatggcttt ttccccgtat ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg
     8341 ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc
     8401 ggctcgggga tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc
     8461 cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg gctgtccctg
     8521 atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa
     8581 caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat
     8641 tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg
     8701 cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc
     8761 tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct
     8821 ttctagggtt aatctagctg catcaggatc atatcgtcgg gtcttttttc cggctcagtc
     8881 atcgcccaag ctggcgctat ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg
     8941 aggttgaagc ggcatggaaa gagtttgccg aggatgactg ctgctgcatt gacgttgagc
     9001 gaaaacgcac gtttaccatg atgattcggg aaggtgtggc catgcacgcc tttaacggtg
     9061 aactgttcgt tcaggccacc tgggatacca gttcgtcgcg gcttttccgg acacagttcc
     9121 ggatggtcag cccgaagcgc atcagcaacc cgaacaatac cggcgacagc cggaactgcc
     9181 gtgccggtgt gcagattaat gacagcggtg cggcgctggg atattacgtc agcgaggacg
     9241 ggtatcctgg ctggatgccg cagaaatgga catggatacc ccgtgagtta cccggcgggc
     9301 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     9361 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     9421 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     9481 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     9541 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     9601 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     9661 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     9721 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     9781 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     9841 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     9901 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     9961 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
    10021 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
    10081 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
    10141 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
    10201 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
    10261 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
    10321 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
    10381 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
    10441 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
    10501 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
    10561 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
    10621 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
    10681 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
    10741 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
    10801 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
    10861 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
    10921 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
    10981 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
    11041 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
    11101 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
    11161 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
    11221 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
    11281 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
    11341 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
    11401 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
    11461 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
    11521 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
    11581 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
    11641 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
    11701 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
    11761 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
    11821 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
    11881 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
    11941 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
    12001 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
    12061 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
    12121 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
    12181 ctcgacacgc tgcagaacac gcagctaga
//
