LOCUS       sequence-346773-       15027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting construct for intergrating a doxycycline (DOX) inducible 
            SAM CRISPR activation complex -2A-GFP to the AAVS1 locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15027)
  AUTHORS   Ma H, de Zwaan E, Guo YE, Cejas P, Thiru P, van de Bunt M, Jeppesen 
            JF, Syamala S, Dall'Agnese A, Abraham BJ, Fu D, Garrett-Engele C, 
            Lee TI, Long HW, Griffith LG, Young RA, Jaenisch R
  TITLE     The nuclear receptor THRB facilitates differentiation of human PSCs 
            into more mature hepatocytes.
  JOURNAL   Cell Stem Cell. 2022 Apr 13. pii: S1934-5909(22)00150-3. doi: 
            10.1016/j.stem.2022.03.015.
  PUBMED    35452598
REFERENCE   2  (bases 1 to 15027)
  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 Stem 
            Cell. 2022 Apr 13. pii: S1934-5909(22)00150-3. doi: 
            10.1016/j.stem.2022.03.015."
COMMENT     Sequence Label: 
            DOX_SAM_CRISPR-activation-2A-GFP-AAVS1-targeting-construct
FEATURES             Location/Qualifiers
     source          1..15027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..804
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    complement(806..839)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    846..871
                     /label=SA
                     /note="splice acceptor site"
     CDS             895..948
                     /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             958..1557
                     /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(958..977)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1454..1474
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1589..1606)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1595..1819
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1825..2204
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2206..2483
                     /label=chicken beta-actin promoter
     intron          2484..3500
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3424..3446
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3508..3527
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3559..4305
                     /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"
     primer_bind     complement(4396..4415)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4461..4516
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4515..4534)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    complement(4865..4898)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     4954..4971
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5088..5109
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5124..5129,5130..5147,5148..5154)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5162..5178
                     /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     5167..5189
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5186..5202
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5186..5202
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5251..5372
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5262..5281)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5316..5335
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5389..6102,6103..6105))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(5392..6102,6103..6105))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5434..5455)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5783..5802
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             complement(6106..6159)
                     /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             complement(6175..6546)
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             complement(6571..7113)
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             complement(7129..7149)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(7204..7590)
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             complement(7594..7647)
                     /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             complement(7654..7803)
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             complement(7828..7848)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(7903..12003)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRRKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQTHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKRTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(12013..12033)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(12135..12159)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    12204..12222
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12240..12258
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12276..12294
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12312..12330
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12348..12366
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12384..12402
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12420..12438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    12471..13307
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     rep_origin      complement(13355..13943)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13435..13454)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14114..14905,14906..14974))
                     /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     14737..14756
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgctttctct gaccagcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt
       61 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
      121 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
      181 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
      241 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
      301 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
      361 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
      421 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
      481 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
      541 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
      601 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
      661 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
      721 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
      781 gtcccctcca ccccacagtg gggcaataac ttcgtatagc atacattata cgaagttata
      841 agcttctgac ctcttctctt cctcccacag ggcctcgaga gatctggcag cggagagggc
      901 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gctcgagatg
      961 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     1021 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     1081 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     1141 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     1201 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     1261 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     1321 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     1381 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     1441 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     1501 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgatct
     1561 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1621 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1681 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1741 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1801 gatgcggtgg gctctatggg tctcgacatt gattattgac tagttattaa tagtaatcaa
     1861 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1921 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1981 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2041 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2101 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2161 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     2221 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     2281 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccrggsg
     2341 gggsggggsg gggsgrgggg sggggsgggg cgaggcggag aggtgcggcg gcagccaatc
     2401 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     2461 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     2521 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     2581 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
     2641 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
     2701 ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg
     2761 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg
     2821 tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga
     2881 acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg
     2941 tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg
     3001 gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc
     3061 aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc
     3121 gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt
     3181 tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga
     3241 aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg
     3301 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc
     3361 ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg
     3421 cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg
     3481 ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt
     3541 ttggcaaaga atgtcgacat gagccggctg gacaagagca aagtgatcaa cagcgccctg
     3601 gaactgctga acggcgtggg catcgagggc ctgaccaccc ggaagctggc ccagaaactg
     3661 ggcgtggaac agcccaccct gtactggcac gtgaagaaca agcgggccct gctggacgcc
     3721 ctgcccatcg agatgctgga ccggcaccac acccacagct gccctctgga aggcgagagc
     3781 tggcaggact tcctgcggaa caacgccaag agctacagat gcgccctgct gagccaccgg
     3841 gacggcgcca aagtgcacct gggcaccaga cccaccgaga agcagtacga gacactggaa
     3901 aaccagctgg ccttcctgtg ccagcagggc ttcagcctgg aaaacgccct gtacgccctg
     3961 agcgccgtgg gccacttcac cctgggctgc gtgctggaag aacaggaaca ccaggtcgcc
     4021 aaagaggaaa gagagacacc caccaccgac agcatgcccc ccctgctgaa gcaggccatc
     4081 gagctgttcg acagacaggg cgccgagccc gccttcctgt tcggcctgga actgatcatc
     4141 tgcggcctcg agaagcagct gaagtgcgag agcggcggac ccaccgacgc cctggacgac
     4201 ttcgacctgg acatgctgcc cgccgatgcc ctggatgatt ttgatctgga tatgctccct
     4261 gctgacgctc tcgatgactt cgatctcgat atgctgccag gctaaaacgc gtgaattcac
     4321 tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca
     4381 caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct
     4441 tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa
     4501 ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa
     4561 tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg
     4621 ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca
     4681 tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc
     4741 tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg
     4801 actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa
     4861 gcttataact tcgtatagca tacattatac gaagttatgc ggccgtcctt tgagtgagct
     4921 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4981 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     5041 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     5101 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     5161 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
     5221 tagctagagg tcgacggtat acagacatga taagatacat tgatgagttt ggacaaacca
     5281 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     5341 ttgtaaccat tataagctgc aataaacaag ttggggtggg cgatcgattt acttgtacag
     5401 ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc
     5461 gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg
     5521 cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac
     5581 gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt
     5641 gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat
     5701 gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc
     5761 ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg
     5821 ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc
     5881 ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg
     5941 cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc
     6001 gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac
     6061 caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcactgggc caggattctc
     6121 ctcgacgtca ccgcatgtta gcagacttcc tctgccctct ccactgccgc tagcggagac
     6181 agtggggtcc ttggctttgg gaggctccga gcctgtcagc agggagatgg tggggtcctc
     6241 ggcgaagccg tccccttcgg agaagtagga gccctctccc agctcaaaca gcaccggcag
     6301 gtcgttgctc ccggtgtcca cggagccggg gtccagcagg aacagcggct gcgctgtgta
     6361 gtgcaccagc tgcttccctg aatccgggct gctgttctct gcctcgggag gcctgggggg
     6421 ctcctgggga gacaggagct cttggatact ggccaggctg ctgtcaaggt caggcaggct
     6481 catgtcgggc acggtcaccg aggggctgaa caggtccagc agggcactgg tgtccacgct
     6541 gaagccgctt ccacctcctc cctgcccact agaggaaatc tgtgacagca gggcactaaa
     6601 gtccatatca gcgatgcttg agaagtcttc atctccggac agcccattag gcaggccgct
     6661 ggttcccagg ggagttggag cggggtcggg gggccgctgg ctgccggtca ccagccgggt
     6721 aatggcttcg gggtactcca tcagcattgg ttcggctgta ctatgagaca tggacacgcc
     6781 ctgattcagc agctgctgaa actcagagtt gtccacggag gccagatctg tgaacactcc
     6841 gggatcggtg ctgttcccca gcagagctcc caggtcctca tcagcgtcga actgcaggtg
     6901 cagcagagct tcactcagag tcccctcgcc ggcctgtgta gacttgggca ctggagcgct
     6961 cagtgactgg ggtggtcctg gggtcagcac aggggctgga gcaggtggct gggccagagg
     7021 caccatagca ctagagggca ccatagtctg ggccagcact ggagcggagc taggggccag
     7081 agccagggcc tggttgctga tctgccctga aggggatcca gcggccgcca ccttcctctt
     7141 tttcttaggt ccgctacctc ctcctccgct tcctcctcct ccgcttccac ctcctccagc
     7201 gctgtagata cctgagttag cggcgatggc ggaagggata ggattaccgt ctttgaggag
     7261 cccctgcatt gccttcacga tgagttcaca gtcagaattg gtagcgaaaa ttgggatagt
     7321 gagctccatg ttcaggtagg acctccaagc ggcgacaggc agttcgactc cgcccactgt
     7381 ctgggtagcc actttgggga cctccacctt gatggtatac tttctcttct gggcactaga
     7441 ctgcctgacg ctgcatgtca ccttgtaggc ctggctccgt gagttggagc tgatccactc
     7501 tgccacccca ttagcgaaat tagaaggagc cactgtcaca tcccctgtcc caccattgtc
     7561 cacgagcacg aactgagtaa agtttgaagc catggggcca gggttctctt ccacgtcgcc
     7621 acatgtcagc aggctgcctc tgccctcgtt aatcagcatg tccaggtcga aatcatcaag
     7681 ggcgtcactg ccgagcatgt cgaggtcaaa gtcatcaagg gcatccgaac caagcatgtc
     7741 aaggtcaaaa tcatcgaggg cgtcacttcc cagcatatcc agatcaaaat cgtccaatgc
     7801 gtcagcccgt ccggatccag cggccgccac cttcctcttt ttcttaggtc cgctacctcc
     7861 tcctccgctt cctcctcctc cgcttccacc tcctccagcg ctgtcgcctc ccagctgaga
     7921 caggtcgatc cgtgtctcgt acaggccggt gatgctctgg tggatcaggg tggcgtccag
     7981 cacctctttg gtgctggtgt acctcttccg gtcgatggtg gtgtcaaagt acttgaaggc
     8041 ggcaggggct cccagattgg tcagggtaaa caggtggatg atattctcgg cctgctctct
     8101 gatgggctta tcccggtgct tgttgtaggc ggacagcact ttgtccagat tagcgtcggc
     8161 caggatcact ctcttggaga actcgctgat ctgctcgatg atctcgtcca ggtagtgctt
     8221 gtgctgttcc acaaacagct gtttctgctc attatcctcg ggggagccct tcagcttctc
     8281 atagtggctg gccaggtaca ggaagttcac atatttggag ggcagggcca gttcgtttcc
     8341 cttctgcagt tcgccggcag aggccagcat tctcttccgg ccgttttcca gctcgaacag
     8401 ggagtactta ggcagcttga tgatcaggtc ctttttcact tctttgtagc ccttggcttc
     8461 cagaaagtcg atgggattct tctcgaagct gcttctttcc atgatggtga tccccagcag
     8521 ctctttcaca ctcttcagtt tcttggactt gcccttttcc actttggcca ccaccagcac
     8581 agaataggcc acggtggggc tgtcgaagcc gccgtacttc ttagggtccc agtccttctt
     8641 tctggcgatc agcttatcgc tgttcctctt gggcaggata gactctttgc tgaagccgcc
     8701 tgtctgcacc tcggtccttt tcacgatatt cacttggggc atgctcagca ctttccgcac
     8761 ggtggcaaaa tcccggccct tatcccacac gatctccccg gtttcgccgt ttgtctcgat
     8821 cagaggccgc ttccggatct cgccgttggc cagggtaatc tcggtcttga aaaagttcat
     8881 gatgttgctg tagaagaagt acttggcggt agccttgccg atttcctgct cgctcttggc
     8941 gatcatcttc cgcacgtcgt acaccttgta gtcgccgtac acgaactcgc tttccagctt
     9001 agggtacttt ttgatcaggg cggttcccac gacggcgttc aggtaggcgt cgtgggcgtg
     9061 gtggtagttg ttgatctcgc gcactttgta aaactggaaa tccttccgga aatcggacac
     9121 cagcttggac ttcagggtga tcactttcac ttcccggatc agcttgtcat tctcgtcgta
     9181 cttagtgttc atccgggagt ccaggatctg tgccacgtgc tttgtgatct gccgggtttc
     9241 caccagctgt ctcttgatga agccggcctt atccagttcg ctcaggccgc ctctctcggc
     9301 cttggtcaga ttgtcgaact ttctctgggt aatcagcttg gcgttcagca gctgccgcca
     9361 gtagttcttc atcttcttca cgacctcttc ggagggcacg ttgtcgctct tgccccggtt
     9421 cttgtcgctt ctggtcagca ccttgttgtc gatggagtcg tccttcagaa agctctgagg
     9481 cacgatagcg tccacatcgt agtcggacag ccggttgatg tccagttcct ggtccacgta
     9541 catatcccgc ccattctgca ggtagtacag gtacagcttc tcgttctgca gctgggtgtt
     9601 ttccacgggg tgttctttca ggatctggct gcccagctct ttgatgccct cttcgatccg
     9661 cttcattctc tcgcggctgt tcttctgtcc cttctgggtg gtctggttct ctctggccat
     9721 ttcgatcacg atgttctcgg gcttgtgccg gcccatcact ttcacgagct cgtccaccac
     9781 cttcactgtc tgcaggatgc ccttcttaat ggcggggctg ccggccagat tggcaatgtg
     9841 ctcgtgcagg ctatcgccct ggccggacac ctgggctttc tggatgtcct ctttaaaggt
     9901 caggctgtcg tcgtggatca gctgcatgaa gtttctgttg gcgaagccgt cggacttcag
     9961 gaaatccagg attgtcttgc cggactgctt gtcccggatg ccgttgatca gcttccggct
    10021 cagcctgccc cagccggtgt atctccgccg cttcagctgc ttcatcactt tgtcgtcgaa
    10081 caggtgggca taggttttca gccgttcctc gatcatctct ctgtcctcaa acagtgtcag
    10141 ggtcagcacg atatcttcca gaatgtcctc gttttcctca ttgtccagga agtccttgtc
    10201 cttgataatt ttcagcagat cgtggtatgt gcccagggag gcgttgaacc gatcttccac
    10261 gccggagatt tccacggagt cgaagcactc gattttcttg aagtagtcct ctttcagctg
    10321 cttcacggtc actttccggt tggtcttgaa cagcaggtcc acgatggcct ttttctgctc
    10381 gccgctcagg aaggcgggct ttctcattcc ctcggtcacg tatttcactt tggtcagctc
    10441 gttatacacg gtgaagtact cgtacagcag gctgtgcttg ggcagcacct tctcgttggg
    10501 caggttctta tcgaagttgg tcatccgctc gatgaagctc tgggcggaag cgcccttgtc
    10561 caccacttcc tcgaagttcc agggggtgat ggtttcctcg ctctttctgg tcatccaggc
    10621 gaatctgctg tttcccctgg ccagagggcc cacgtagtag gggatgcgga aggtcaggat
    10681 cttctcgatc ttttcccggt tgtccttcag gaatgggtaa aaatcttcct gccgccgcag
    10741 aatggcgtgc agctctccca ggtgggtctg gtgggggatg ctgccgttgt cgaaggtccg
    10801 ctgcttccgc agcaggtcct ctctgttcag cttcacgagc agttcctcgg tgccgtccat
    10861 cttttccagg atgggcttga tgaacttgta gaactcttcc tggctggctc cgccgtcaat
    10921 gtagccggcg tagccgttct tgctctggtc gaagaaaatc tctttgtact tctcaggcag
    10981 ctgctgccgc acgagagctt tcagcagggt caggtcctgg tggtgctcgt cgtatctctt
    11041 gatcatagag gcgctcaggg gggccttggt gatctcggtg ttcactctca ggatgtcgct
    11101 cagcaggatg gcgtcggaca ggttcttggc ggccagaaac aggtcggcgt actggtcgcc
    11161 gatctgggcc agcaggttgt ccaggtcgtc gtcgtaggtg tccttgctca gctgcagttt
    11221 ggcatcctcg gccaggtcga agttgctctt gaagttgggg gtcaggccca ggctcagggc
    11281 aatcaggttg ccgaacaggc cattcttctt ctcgccgggc agctgggcga tcagattttc
    11341 cagccgtctg ctcttgctca gtctggcaga caggatggcc ttggcgtcca cgccgctggc
    11401 gttgatgggg ttttcctcga acagctggtt gtaggtctgc accagctgga tgaacagctt
    11461 gtccacgtcg ctgttgtcgg ggttcaggtc gccctcgatc aggaagtggc cccggaactt
    11521 gatcatgtgg gccagggcca gatagatcag ccgcaggtcg gccttgtcgg tgctgtccac
    11581 cagtttcctt ctcaggtggt agatggtggg gtacttctcg tggtaggcca cctcgtccac
    11641 gatgttgccg aagatggggt gccgctcgtg cttcttatcc tcttccacca ggaaggactc
    11701 ttccagtctg tggaagaagc tgtcgtccac cttggccatc tcgttgctga agatctcttg
    11761 cagatagcag atccggttct tccgtctggt gtatcttctt ctggcggttc tcttcagccg
    11821 ggtggcctcg gctgtttcgc cgctgtcgaa cagcagggct ccgatcaggt tcttcttgat
    11881 gctgtgccgg tcggtgttgc ccagcacctt gaatttcttg ctgggcacct tgtactcgtc
    11941 ggtgatcacg gcccagccca cagagttggt gccgatggcc aggccgatgc tgtacttctt
    12001 gtcgctggcc tccacctttc tcttcttctt ggggctcatg gtggtaccta agttaattaa
    12061 ccttaagttt acgagggtag gaagtggtac ggaaagttgg tataagacaa aagtgttgtg
    12121 gaattgctcc aggcgatctg acggttcact aaacgagctc tgcttttata ggcgcccacc
    12181 gtacacgcct aaagcttata cgttctctat cactgatagg gagtaaactg gatatacgtt
    12241 ctctatcact gatagggagt aaactgtaga tacgttctct atcactgata gggagtaaac
    12301 tggtcatacg ttctctatca ctgataggga gtaaactcct tatacgttct ctatcactga
    12361 tagggagtaa agtctgcata cgttctctat cactgatagg gagtaaactc ttcatacgtt
    12421 ctctatcact gatagggagt aaaccactgt ggggtggaga tatcagcttt actagggaca
    12481 ggattggtga cagaaaagcc ccatccttag gcctcctcct tcctagtctc ctgatattgg
    12541 gtctaacccc cacctcctgt taggcagatt ccttatctgg tgacacaccc ccatttcctg
    12601 gagccatctc tctccttgcc agaacctcta aggtttgctt acgatggagc cagagaggat
    12661 cctgggaggg agagcttggc agggggtggg agggaagggg gggatgcgtg acctgcccgg
    12721 ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg agctgctctg acgcggctgt
    12781 ctggtgcgtt tcactgatcc tggtgctgca gcttccttac acttcccaag aggagaagca
    12841 gtttggaaaa acaaaatcag aataagttgg tcctgagttc taactttggc tcttcacctt
    12901 tctagtcccc aatttatatt gttcctccgt gcgtcagttt tacctgtgag ataaggccag
    12961 tagccagccc cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt ggaaaactcc
    13021 ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct actccctttc
    13081 tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca tattcctccg
    13141 cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt tgagtccttg
    13201 gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac catctcatgc
    13261 ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctctaag ggcgaattcg
    13321 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
    13381 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
    13441 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
    13501 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
    13561 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
    13621 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
    13681 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
    13741 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
    13801 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
    13861 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
    13921 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
    13981 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
    14041 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
    14101 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
    14161 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
    14221 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
    14281 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
    14341 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
    14401 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
    14461 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
    14521 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
    14581 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
    14641 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
    14701 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
    14761 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
    14821 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
    14881 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
    14941 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
    15001 gcatttatca gggttgaatt cgccctt
//
