LOCUS       sequence-233059-       13587 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Stably express BE3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13587)
  AUTHORS   Li Q, Li Y, Yang S, Huang S, Yan M, Ding Y, Tang W, Lou X, Yin Q, 
            Sun Z, Lu L, Shi H, Wang H, Chen Y, Li J
  TITLE     CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 
            amino acids that are critical for primordial germ cell development.
  JOURNAL   Nat Cell Biol. 2018 Oct 1. pii: 10.1038/s41556-018-0202-4. doi: 
            10.1038/s41556-018-0202-4.
  PUBMED    30275529
REFERENCE   2  (bases 1 to 13587)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Cell 
            Biol. 2018 Oct 1. pii: 10.1038/s41556-018-0202-4. doi: 
            10.1038/s41556-018-0202-4."
COMMENT     Sequence Label: PB-BE3-2NLS
FEATURES             Location/Qualifiers
     source          1..13587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   892..954
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        2038..2417
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2420..2695
                     /label=chicken beta-actin promoter
     intron          2696..3708
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3632..3654
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3716..3735
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3768..4442
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3768..4442
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(3898..3916)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4166..4185
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4360..4383
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             4446..4499
                     /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             4500..4565
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4572..4592
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4614..5300
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             join(5349..8747,8748..8750,8751..9347,9348,9349..9351,
                     9352..9353,9354..9356,9357..9449)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(5349..5372,5373..5375,5376..9449)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             9462..9710
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             9723..9743
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9752..9769
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(9896..9915)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    9961..10016
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(10015..10034)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(10377..10393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    10401..10417
                     /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(10425..10431,10432..10449,10450..10455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10470..10491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   11192..11226
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11399..11419)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(11399..11417)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(11438..11454)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    11462..11478
                     /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(11486..11492,11493..11510,11511..11516))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11531..11552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11669..11686)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11840..12428)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11920..11939)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12599..13390,13391..13459))
                     /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     13222..13241
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13460..13564)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggccgc ccggtttatc gttaatatgg atcaatttga acagttgatt aacgtgtctc
      721 tgctcaagtc tttgatcaaa acgcaaatcg acgaaaatgt gtcggacaat atcaagtcga
      781 tgagcgaaaa actaaaaagg ctagaatacg acaatctcac agacagcgtt gagatatacg
      841 gtattcacga cagcaggctg aataataaaa aaattagaaa ctattattta accctagaaa
      901 gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt
      961 atcggtctgt atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt
     1021 tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta
     1081 aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttaa acattctctc
     1141 ttttacaaaa ataaacttat tttgtacttt aaaaacagtc atgttgtatt ataaaataag
     1201 taattagctt aacttataca taatagaaac aaattatact tattagtcag tcagaaacaa
     1261 ctttggcaca tatcaatatt atgctctcga caaataactt ttttgcattt tttgcacgat
     1321 gcatttgcct ttcgccttat tttagagggg cagtaagtac agtaagtacg ttttttcatt
     1381 actggctctt cagtactgtc atctgatgta ccaggcactt catttggcaa aatattagag
     1441 atattatcgc gcaaatatct cttcaaagta ggagcttcta aacgcttacg cataaacgat
     1501 gacgtcaggc tcatgtaaag gtttctcata aattttttgc gactttgaac cttttctccc
     1561 ttgctactga cattatggct gtatataata aaagaattta tgcaggcaat gtttatcatt
     1621 ccgtacaata atgccatagg ccacctattc gtcttcctac tgcaggtcat cacagaacac
     1681 atttggtcta gcgtgtccac tccgccttta gtttgattat aatacataac catttgcggt
     1741 ttaccggtac tttcgttgat agaagcatcc tcatcacaag atgataataa gtataccatc
     1801 ttagctggct tcggtttata tgagacgaga gtaaggggtc cgtcaaaaca aaacatcgat
     1861 gttcccactg gcctggagcg actgtttttc agtacttccg gtatctcgcg tttgtttgat
     1921 cgcacggttc ccacaatggt taattcgagc tcgcccaaac cgggcgcgcc gggcatttaa
     1981 atggaattcg atgccctata tatggatcca attgcaatga tcatcatgac agatctcgac
     2041 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     2101 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     2161 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2221 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     2281 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2341 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2401 tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
     2461 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
     2521 ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg
     2581 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
     2641 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
     2701 cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc cgccccggct
     2761 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     2821 taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg aaagccttaa
     2881 agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg tgcgtgtgtg
     2941 tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc gctgcgggcg
     3001 cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg gggcggtgcc
     3061 ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg
     3121 ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca cccccctccc
     3181 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
     3241 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
     3301 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
     3361 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
     3421 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
     3481 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
     3541 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     3601 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     3661 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     3721 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgatccgatg gcctcctccg
     3781 aggacgtcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc
     3841 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     3901 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     3961 tccagtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     4021 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     4081 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     4141 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     4201 cctccaccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagatgaggc
     4261 tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa gaccacctac atggccaaga
     4321 agcccgtgca gctgcccggc gcctacaaga ccgacatcaa gctggacatc acctcccaca
     4381 acgaggacta caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggcg
     4441 cccccgaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag
     4501 actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg
     4561 ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtcacg cgtatgagct
     4621 cagagactgg cccagtggct gtggacccca cattgagacg gcggatcgag ccccatgagt
     4681 ttgaggtatt cttcgatccg agagagctcc gcaaggagac ctgcctgctt tacgaaatta
     4741 attggggggg ccggcactcc atttggcgac atacatcaca gaacactaac aagcacgtcg
     4801 aagtcaactt catcgagaag ttcacgacag aaagatattt ctgtccgaac acaaggtgca
     4861 gcattacctg gtttctcagc tggagcccat gcggcgaatg tagtagggcc atcactgaat
     4921 tcctgtcaag gtatccccac gtcactctgt ttatttacat cgcaaggctg taccaccacg
     4981 ctgacccccg caatcgacaa ggcctgcggg atttgatctc ttcaggtgtg actatccaaa
     5041 ttatgactga gcaggagtca ggatactgct ggagaaactt tgtgaattat agcccgagta
     5101 atgaagccca ctggcctagg tatccccatc tgtgggtacg actgtacgtt cttgaactgt
     5161 actgcatcat actgggcctg cctccttgtc tcaacattct gagaaggaag cagccacagc
     5221 tgacattctt taccatcgct cttcagtctt gtcattacca gcgactgccc ccacacattc
     5281 tctgggccac cgggttgaaa agcggcagcg agactcccgg gacctcagag tccgccacac
     5341 ccgaaagtga taaaaagtat tctattggtt tagccatcgg cactaattcc gttggatggg
     5401 ctgtcataac cgatgaatac aaagtacctt caaagaaatt taaggtgttg gggaacacag
     5461 accgtcattc gattaaaaag aatcttatcg gtgccctcct attcgatagt ggcgaaacgg
     5521 cagaggcgac tcgcctgaaa cgaaccgctc ggagaaggta tacacgtcgc aagaaccgaa
     5581 tatgttactt acaagaaatt tttagcaatg agatggccaa agttgacgat tctttctttc
     5641 accgtttgga agagtccttc cttgtcgaag aggacaagaa acatgaacgg caccccatct
     5701 ttggaaacat agtagatgag gtggcatatc atgaaaagta cccaacgatt tatcacctca
     5761 gaaaaaagct agttgactca actgataaag cggacctgag gttaatctac ttggctcttg
     5821 cccatatgat aaagttccgt gggcactttc tcattgaggg tgatctaaat ccggacaact
     5881 cggatgtcga caaactgttc atccagttag tacaaaccta taatcagttg tttgaagaga
     5941 accctataaa tgcaagtggc gtggatgcga aggctattct tagcgcccgc ctctctaaat
     6001 cccgacggct agaaaacctg atcgcacaat tacccggaga gaagaaaaat gggttgttcg
     6061 gtaaccttat agcgctctca ctaggcctga caccaaattt taagtcgaac ttcgacttag
     6121 ctgaagatgc caaattgcag cttagtaagg acacgtacga tgacgatctc gacaatctac
     6181 tggcacaaat tggagatcag tatgcggact tatttttggc tgccaaaaac cttagcgatg
     6241 caatcctcct atctgacata ctgagagtta atactgagat taccaaggcg ccgttatccg
     6301 cttcaatgat caaaaggtac gatgaacatc accaagactt gacacttctc aaggccctag
     6361 tccgtcagca actgcctgag aaatataagg aaatattctt tgatcagtcg aaaaacgggt
     6421 acgcaggtta tattgacggc ggagcgagtc aagaggaatt ctacaagttt atcaaaccca
     6481 tattagagaa gatggatggg acggaagagt tgcttgtaaa actcaatcgc gaagatctac
     6541 tgcgaaagca gcggactttc gacaacggta gcattccaca tcaaatccac ttaggcgaat
     6601 tgcatgctat acttagaagg caggaggatt tttatccgtt cctcaaagac aatcgtgaaa
     6661 agattgagaa aatcctaacc tttcgcatac cttactatgt gggacccctg gcccgaggga
     6721 actctcggtt cgcatggatg acaagaaagt ccgaagaaac gattactcca tggaattttg
     6781 aggaagttgt cgataaaggt gcgtcagctc aatcgttcat cgagaggatg accaactttg
     6841 acaagaattt accgaacgaa aaagtattgc ctaagcacag tttactttac gagtatttca
     6901 cagtgtacaa tgaactcacg aaagttaagt atgtcactga gggcatgcgt aaacccgcct
     6961 ttctaagcgg agaacagaag aaagcaatag tagatctgtt attcaagacc aaccgcaaag
     7021 tgacagttaa gcaattgaaa gaggactact ttaagaaaat tgaatgcttc gattctgtcg
     7081 agatctccgg ggtagaagat cgatttaatg cgtcacttgg tacgtatcat gacctcctaa
     7141 agataattaa agataaggac ttcctggata acgaagagaa tgaagatatc ttagaagata
     7201 tagtgttgac tcttaccctc tttgaagatc gggaaatgat tgaggaaaga ctaaaaacat
     7261 acgctcacct gttcgacgat aaggttatga aacagttaaa gaggcgtcgc tatacgggct
     7321 ggggacgatt gtcgcggaaa cttatcaacg ggataagaga caagcaaagt ggtaaaacta
     7381 ttctcgattt tctaaagagc gacggcttcg ccaataggaa ctttatgcag ctgatccatg
     7441 atgactcttt aaccttcaaa gaggatatac aaaaggcaca ggtttccgga caaggggact
     7501 cattgcacga acatattgcg aatcttgctg gttcgccagc catcaaaaag ggcatactcc
     7561 agacagtcaa agtagtggat gagctagtta aggtcatggg acgtcacaaa ccggaaaaca
     7621 ttgtaatcga gatggcacgc gaaaatcaaa cgactcagaa ggggcaaaaa aacagtcgag
     7681 agcggatgaa gagaatagaa gagggtatta aagaactggg cagccagatc ttaaaggagc
     7741 atcctgtgga aaatacccaa ttgcagaacg agaaacttta cctctattac ctacaaaatg
     7801 gaagggacat gtatgttgat caggaactgg acataaaccg tttatctgat tacgacgtcg
     7861 atcacattgt accccaatcc tttttgaagg acgattcaat cgacaataaa gtgcttacac
     7921 gctcggataa gaaccgaggg aaaagtgaca atgttccaag cgaggaagtc gtaaagaaaa
     7981 tgaagaacta ttggcggcag ctcctaaatg cgaaactgat aacgcaaaga aagttcgata
     8041 acttaactaa agctgagagg ggtggcttgt ctgaacttga caaggccgga tttattaaac
     8101 gtcagctcgt ggaaacccgc caaatcacaa agcatgttgc acagatacta gattcccgaa
     8161 tgaatacgaa atacgacgag aacgataagc tgattcggga agtcaaagta atcactttaa
     8221 agtcaaaatt ggtgtcggac ttcagaaagg attttcaatt ctataaagtt agggagataa
     8281 ataactacca ccatgcgcac gacgcttatc ttaatgccgt cgtagggacc gcactcatta
     8341 agaaataccc gaagctagaa agtgagtttg tgtatggtga ttacaaagtt tatgacgtcc
     8401 gtaagatgat cgcgaaaagc gaacaggaga taggcaaggc tacagccaaa tacttctttt
     8461 attctaacat tatgaatttc tttaagacgg aaatcactct ggcaaacgga gagatacgca
     8521 aacgaccttt aattgaaacc aatggggaga caggtgaaat cgtatgggat aagggccggg
     8581 acttcgcgac ggtgagaaaa gttttgtcca tgccccaagt caacatagta aagaaaactg
     8641 aggtgcagac cggagggttt tcaaaggaat cgattcttcc aaaaaggaat agtgataagc
     8701 tcatcgctcg taaaaaggac tgggacccga aaaagtacgg tggcttcgat agccctacag
     8761 ttgcctattc tgtcctagta gtggcaaaag ttgagaaggg aaaatccaag aaactgaagt
     8821 cagtcaaaga attattgggg ataacgatta tggagcgctc gtcttttgaa aagaacccca
     8881 tcgacttcct tgaggcgaaa ggttacaagg aagtaaaaaa ggatctcata attaaactac
     8941 caaagtatag tctgtttgag ttagaaaatg gccgaaaacg gatgttggct agcgccggag
     9001 agcttcaaaa ggggaacgaa ctcgcactac cgtctaaata cgtgaatttc ctgtatttag
     9061 cgtcccatta cgagaagttg aaaggttcac ctgaagataa cgaacagaag caactttttg
     9121 ttgagcagca caaacattat ctcgacgaaa tcatagagca aatttcggaa ttcagtaaga
     9181 gagtcatcct agctgatgcc aatctggaca aagtattaag cgcatacaac aagcacaggg
     9241 ataaacccat acgtgagcag gcggaaaata ttatccattt gtttactctt accaacctcg
     9301 gcgctccagc cgcattcaag tattttgaca caacgataga tcgcaaacga tacacttcta
     9361 ccaaggaggt gctagacgcg acactgattc accaatccat cacgggatta tatgaaactc
     9421 ggatagattt gtcacagctt gggggtgact ctggtggttc tactaatctg tcagatatta
     9481 ttgaaaagga gaccggtaag caactggtta tccaggaatc catcctcatg ctcccagagg
     9541 aggtggaaga agtcattggg aacaagccgg aaagcgatat actcgtgcac accgcctacg
     9601 acgagagcac cgacgagaat gtcatgcttc tgactagcga cgcccctgaa tacaagcctt
     9661 gggctctggt catacaggat agcaacggtg agaacaagat taagatgctc tctggtggtt
     9721 ctcccaagaa gaagaggaaa gtctaaccgg tcatcatcac catcaccatt gagtttaaac
     9781 ccgctgatca gcacgcgtgc tagcttcgaa gctaattcac tcctcaggtg caggctgcct
     9841 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     9901 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     9961 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
    10021 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
    10081 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc
    10141 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag
    10201 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt
    10261 ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg
    10321 tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca
    10381 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
    10441 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
    10501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatcga tctgacaatg
    10561 ttcagtgcag agactcggct acgcctcgtg gactttgaag ttgaccaaca atgtttattc
    10621 ttacctctaa tagtcctctg tggcaaggtc aagattctgt tagaagccaa tgaagaacct
    10681 ggttgttcaa taacattttg ttcgtctaat atttcactac cgcttgacgt tggctgcact
    10741 tcatgtacct catctataaa cgcttcttct gtatcgctct ggacgtcatc ttcacttacg
    10801 tgatctgata tttcactgtc agaatcctca ccaacaagct cgtcatcgct ttgcagaaga
    10861 gcagagagga tatgctcatc gtctaaagaa ctacccattt tattatatat tagtcacgat
    10921 atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca
    10981 atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt
    11041 tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc
    11101 tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta
    11161 tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt
    11221 ctagggttaa aaaagatttg cgctttactc gacctaaact ttaaacacgt catagaatct
    11281 tcgtttgaca aaaaccacat tgtggccaag ctgtgtgacg cgacgcgcgc taaagaatgg
    11341 caaaccaagt cgcgcgagcg tcgacctcga gggggggccc ggtacccagc ttttgttccc
    11401 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
    11461 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
    11521 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
    11581 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
    11641 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
    11701 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
    11761 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
    11821 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
    11881 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
    11941 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
    12001 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
    12061 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
    12121 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
    12181 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
    12241 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
    12301 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
    12361 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
    12421 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
    12481 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
    12541 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
    12601 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
    12661 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
    12721 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
    12781 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
    12841 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
    12901 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
    12961 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
    13021 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
    13081 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
    13141 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
    13201 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
    13261 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
    13321 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
    13381 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
    13441 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
    13501 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
    13561 cgcgcacatt tccccgaaaa gtgccac
//
