LOCUS       pLV-EF1A-CD19.28        8879 bp DNA     circular SYN 23-MAR-2023
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8879)
  AUTHORS   Michele Bernasconi
  TITLE     Direct Submission
  JOURNAL   Exported Mar 30, 2023 from SnapGene 6.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8879
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     promoter        1..229
                     /label=RSV  promoter
                     /note="/application_notes=Strong promoter; drives 
                     transcriptionof viral RNA in packaging cells."
                     /note="/description=Rous sarcoma virus enhancer/promoter"
                     /note="/full_name=Rous sarcoma virus (RSV) 
                     enhancer/promoter"
                     /note="/official_designation=RSV"
                     /note="/uuid=3f2ddca7f0cb4015b6abcec8a25c834e"
                     /note="color: #ef6cdf; direction: RIGHT"
     LTR             230..410
                     /label=5' LTR-Delta-U3
                     /note="/application_notes=Allows transcription of viral RNA
                     andits packaging into virus."
                     /note="/description=Truncated HIV-1 5' long terminal 
                     repeat"
                     /note="/full_name=HIV-1 truncated 5' LTR"
                     /note="/official_designation=Delta5' LTR"
                     /note="/uuid=44156a3083604321be44066133e8ecb9"
                     /note="color: #c54b7c; direction: RIGHT"
     misc_feature    521..565
                     /label=Psi
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Allows packaging of viral RNA 
                     intovirus."
                     /note="/description=HIV-1 packaging signal"
                     /note="/full_name=HIV-1 psi packaging signal"
                     /note="/official_designation=Psi"
                     /note="/uuid=284899413ff943a99f8210a11ad8c54e"
                     /note="color: #d05c0a; direction: RIGHT"
     misc_feature    566..930
                     /locus_tag="gag"
                     /label=gag
                     /note="color: #a6acb3"
     misc_feature    1075..1308
                     /label=RRE
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Rev protein binding site that 
                     allowsRev-dependent nuclear export of viral RNA during 
                     viral packaging."
                     /note="/description=HIV-1 Rev response element"
                     /note="/full_name=HIV-1 Rev response element"
                     /note="/official_designation=RRE"
                     /note="/uuid=df07099e49674cf3aba3661b54e28bd7"
                     /note="color: #fd3434; direction: RIGHT"
     CDS             1493..1537
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /note="color: #cc99b2; direction: RIGHT"
     misc_feature    1797..1802
                     /label=NheI
                     /note="color: #a6acb3"
     misc_feature    1803..1920
                     /label=cPPT
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Facilitates the nuclear import of
                     HIV-1cDNA through a central DNA flap."
                     /note="/description=Central polypurine tract"
                     /note="/full_name=Central polypurine tract"
                     /note="/official_designation=cPPT"
                     /note="/uuid=f7cb29985f9f41a7a5924d3462266f42"
                     /note="color: #46c6ef; direction: RIGHT"
     protein_bind    1938..1958
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
                     /note="color: #31849b; direction: RIGHT"
     misc_feature    1959..1964
                     /label=XbaI
                     /note="color: #a6acb3"
     promoter        1965..2176
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    2040..2045
                     /label=Kozak
                     /note="color: #a6acb3"
     primer_bind     2065..2084
                     /label=pSL-1F
                     /note="color: #------; direction: RIGHT"
     primer_bind     2133..2208
                     /label=CPF01
                     /note="color: black; sequence: 
                     ccgtgaacgttctttttcgcaacgggtttgccgccagaacacagggatcccaagtttgt
                     acaaaaaagcaggctgc; added: 2020-10-29"
     misc_feature    2177..2182
                     /label=BamHI
                     /note="color: #a6acb3"
     protein_bind    2182..2206
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
                     /note="color: #------"
     misc_feature    2207..2212
                     /label=Kozak
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Facilitates translation 
                     initiation ofATG start codon downstream of the Kozak 
                     sequence."
                     /note="/description=Kozak translation initiation sequence"
                     /note="/full_name=Kozak"
                     /note="/official_designation=Kozak"
                     /note="/uuid=5f18b25a1234443494a618363f09f052"
                     /note="color: #c0c0c0"
     CDS             2213..2266
                     /codon_start=1
                     /gene="CD28"
                     /locus_tag="T-cell-specific surface glycoprotein 
                     CD28isoform 1 precursor"
                     /product="T-cell-specific surface glycoprotein CD28 
                     isoform1 precursor"
                     /label=Signal peptide CD28
                     /label=CD28 signaling peptide
                     /label=T-cell-specific surface glycoprotein CD28 isofo
                     /label=T-cell-specific surface glycoprotein CD28 isoform 1 
                     precursor
                     /note="isoform 1 precursor is encoded by transcript 
                     variant1; CD28 antigen"
                     /note="color: #993366"
                     /db_xref="GI:5453611"
                     /db_xref="CCDS:CCDS2361.1"
                     /db_xref="GeneID:940"
                     /db_xref="HGNC:HGNC:1653"
                     /db_xref="HPRD:01727"
                     /db_xref="MIM:186760"
                     /protein_id="NP_006130.1"
                     /translation="MLRLLLALNLFPSIQVTG"
     CDS             2267..2272
                     /codon_start=1
                     /label=XhoI
                     /note="color: #993366"
                     /translation="LE"
     CDS             2273..2302
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #00ff00"
                     /translation="EQKLISEEDL"
     CDS             2303..2332
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #00ff00"
                     /translation="EQKLISEEDL"
     primer_bind     complement(2311..2373)
                     /label=CPR01
                     /note="color: black; sequence: 
                     gtctgtgtcatctggatgtcggCGTCTCcGTCGACCTCGAGcagatcttcttctgaaat
                     gagc; added: 2020-10-29"
     primer_bind     2317..2380
                     /label=CPF02
                     /note="color: black; sequence: 
                     ttcagaagaagatctgCTCGAGGTCGACgGAGACGccgacatccagatgacacagacta
                     catcc; added: 2020-11-19"
     CDS             2333..2338
                     /codon_start=1
                     /label=XhoI
                     /note="color: #c0c0c0"
                     /translation="LE"
     CDS             2339..2344
                     /codon_start=1
                     /label=SalI
                     /note="color: #c0c0c0"
                     /translation="VD"
     CDS             2345..2353
                     /codon_start=1
                     /label=Esp3I filler
                     /note="color: #c0c0c0"
                     /translation="GDA"
     CDS             2354..3076
                     /codon_start=1
                     /label=FMC63 scFv
                     /label=FMC63-28Z receptor protein
                     /note="color: #ffcc00"
                     /translation="DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK
                     LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL
                     EITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR
                     QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAK
                     HYYYGGSYAMDYWGQGTSV"
     misc_feature    2435..2455
                     /label=IDT optimization
                     /note="color: #------"
     primer_bind     3062..3119
                     /label=CPF03b
                     /note="color: black; sequence: 
                     caaggaacctcagtcaccgtctccGGTACCATTGAAGTTATGTATCCTCCTCCTTACC;
                      added: 2020-11-19"
     primer_bind     complement(3062..3114)
                     /label=CPR02b
                     /note="color: black; sequence: 
                     GGAGGAGGATACATAACTTCAATGGTACCggagacggtgactgaggttccttg; 
                     added: 2020-11-19"
     primer_bind     complement(3062..3092)
                     /label=CPR02a
                     /note="color: black; sequence: 
                     ggccgaggagcgggcgttgtggtGGTACCggagacggtgactgaggttccttg; 
                     added: 2020-10-30"
     CDS             3077..3085
                     /codon_start=1
                     /label=Esp3I filler 2
                     /note="color: #c0c0c0"
                     /translation="TVS"
     CDS             3086..3091
                     /codon_start=1
                     /label=KpnI
                     /note="color: #c0c0c0"
                     /translation="GT"
     gene            3092..3293
                     /gene="CD28"
                     /gene_synonym="Tp44"
                     /label=CD28
                     /note="CD28 molecule"
                     /note="color: #a6acb3; direction: RIGHT"
                     /db_xref="GeneID:940"
                     /db_xref="HGNC:HGNC:1653"
                     /db_xref="MIM:186760"
     mat_peptide     3092..3293
                     /gene="CD28"
                     /gene_synonym="Tp44"
                     /product="T-cell-specific surface glycoprotein CD28isoform 
                     1"
                     /note="color: #cc99b2"
     CDS             3092..3196
                     /codon_start=1
                     /label=CD28 hinge
                     /note="color: #ccffcc"
                     /translation="IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG"
     exon            3092..3161
                     /gene="CD28"
                     /gene_synonym="Tp44"
                     /note="color: #708090"
                     /inference="alignment:same species:2.1.0"
     misc_feature    3137..3139
                     /gene="CD28"
                     /gene_synonym="Tp44"
                     /note="N-linked (GlcNAc...) asparagine. 
                     /evidence=ECO:0000269|PubMed:19349973; propagated from 
                     UniProtKB/Swiss-Prot (P10747.1); glycosylation site"
                     /note="color: #a6acb3"
     exon            3162..3293
                     /gene="CD28"
                     /gene_synonym="Tp44"
                     /note="This feature has 2 segments:
                        1: 3162 .. 3286 / #708090
                        2: 3287 .. 3293 / #708090"
                     /inference="alignment:same species:2.1.0"
     CDS             3197..3289
                     /codon_start=1
                     /locus_tag="CD28TM"
                     /label=CD28TM
                     /note="color: #ccffcc"
                     /translation="PSKPFWVLVVVGGVLACYSLLVTVAFIIFWV"
     misc_feature    3209..3289
                     /gene="CD28"
                     /gene_synonym="Tp44"
                     /label=TM
                     /note="propagated from UniProtKB/Swiss-Prot (P10747.1); 
                     transmembrane region"
                     /note="color: #------"
     primer_bind     complement(3266..3312)
                     /label=CPR03d
                     /note="color: black; sequence: 
                     agtttctttctgccccgtttcctcacccagaaaataataaaggccac; added: 
                     2020-11-19"
     primer_bind     complement(3266..3293)
                     /label=CPR03c
                     /note="color: black; sequence: 
                     tgcaggagcctgctcctcttactcctcacccagaaaataataaaggccac; added: 
                     2020-11-19"
     primer_bind     3273..3314
                     /label=CPF04d
                     /note="color: black; sequence: 
                     ttattattttctgggtgaggaaacggggcagaaagaaactcc; added: 
                     2020-10-29"
     CDS             3290..3292
                     /codon_start=1
                     
                     /note="color: #33cccc"
                     /translation="R"
     CDS             3293..3418
                     /codon_start=1
                     /product="41BB CSD"
                     /label=41BB
                     /note="color: #0000ff"
                     /translation="KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL"
     primer_bind     complement(3418..3441)
                     /label=CPR03c
                     /note="color: black; sequence: 
                     tgcaggagcctgctcctcttactcctcacccagaaaataataaaggccac; added: 
                     2020-11-19"
     CDS             3419..3538
                     /codon_start=1
                     /label=CD28
                     /note="color: #33cccc"
                     /translation="SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS"
     CDS             3539..3544
                     /codon_start=1
                     /label=Kpn2I
                     /note="color: #c0c0c0"
                     /translation="SG"
     CDS             3545..3886
                     /codon_start=1
                     /label=CD3z isoform 1 - no Esp3I
                     /note="color: #ff0000"
                     /translation="LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
                     MGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD
                     ALHMQALPPR"
     misc_feature    3661
                     /label=a->g
                     /note="a to g mutated to eliminate Esp3I site"
                     /note="color: #ffff00"
     primer_bind     complement(3877..3942)
                     /label=CPR04
                     /note="color: black; sequence: 
                     tcttccacgtcgcctgcttgctttaacagagagaagttcgtggccgtacgTCCGGAgcg
                     agggggc; added: 2020-10-29"
     CDS             3887..3892
                     /codon_start=1
                     /label=Kpn2I
                     /note="color: #c0c0c0"
                     /translation="SG"
     CDS             3893..3898
                     /codon_start=1
                     /label=BsiWI
                     /note="color: #c0c0c0"
                     /translation="RT"
     misc_feature    3898..4681
                     /label={2nd scFv-28TM-CD28-BB-CD3z CAR}
                     /label=nonstandard type:  ORF
                     /note="/application_notes="
                     /note="/official_designation={2nd scFv-28TM-CD28-BB-CD3z 
                     CAR}"
                     /note="/uuid=a7af8ce6d40a4636ba582c49665c9db3"
                     /note="color: #028a02; direction: RIGHT"
     CDS             3899..3955
                     /codon_start=1
                     /locus_tag="P2A"
                     /label=P2A Signal_peptide
                     /label=nonstandard type: Signal_peptide
                     /note="color: #ff9900"
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3923..3944)
                     /label=pSL-2R
                     /note="Sequencing with this primer did not work. 28.8.2019
                     Probably the GGG stretch is too long...
                     "
                     /note="color: #------; direction: LEFT"
     primer_bind     complement(3923..3944)
                     /label=pSL-2R
                     /note="color: black; sequence: tttcttccacgtcgcctgcttg; 
                     added: 2023-03-23"
     CDS             3953..4672
                     /codon_start=1
                     /label=EGFP after P2A
                     /note="color: #------"
                     /translation="PVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3956..4672
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="This feature has 2 segments:
                        1: 3956 .. 3958 / #05fd14 / 1a
                        2: 3959 .. 4672 / #05fd14"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    4064..4069
                     /label=Kozak
                     /note="color: #a6acb3"
     protein_bind    complement(4682..4706)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
                     /note="color: #31849b; direction: LEFT"
     misc_feature    4720..5317
                     /label=WPRE
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Enhances virus stability in 
                     packagingcells, leading to higher titer of packaged virus; 
                     enhances higher expression of transgenes."
                     /note="/description=Woodchuck hepatitis virus 
                     posttranscriptionalregulatory element"
                     /note="/full_name=Woodchuck hepatitis virus 
                     posttranscriptionalregulatory element"
                     /note="/official_designation=WPRE"
                     /note="/uuid=0aedff8308474b369c622433538d7836"
                     /note="color: #e4b930; direction: RIGHT"
     CDS             complement(5195..5206)
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2; direction: LEFT
                     Cleavage site after base 5194"
     LTR             5399..5633
                     /label=3' LTR-Delta-U3
                     /note="/application_notes=Allows packaging of viral RNA 
                     intovirus; self-inactivates the 5' LTR by a copying 
                     mechanism during viral genome integration; contains 
                     polyadenylation signal for transcription termination."
                     /note="/description=Truncated HIV-1 3' long terminal 
                     repeat"
                     /note="/full_name=HIV-1 truncated 3' LTR"
                     /note="/official_designation=DeltaU3/3' LTR"
                     /note="/uuid=c958755414c34372b909e20e81ebcef6"
                     /note="color: #05696d; direction: RIGHT"
     polyA_signal    5706..5840
                     /label=SV40 early pA
                     /note="/application_notes=Allows transcription termination 
                     andpolyadenylation of mRNA transcribed by Pol II RNA 
                     polymerase."
                     /note="/description=Simian virus 40 early polyadenylation 
                     signal"
                     /note="/full_name=SV40 early polyadenation signal"
                     /note="/official_designation=SV40 early pA"
                     /note="/uuid=2fcb13a62e704d8095b188d9625a6a94"
                     /note="color: #f54c6b; direction: RIGHT"
     rep_origin      5866..6001
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     promoter        complement(6022..6040)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(6050..6066)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     rep_origin      6208..6663
                     /locus_tag="F1 ori"
                     /label=F1 ori
                     /note="color: #ffff00"
     misc_feature    6665..6670
                     /label=Kozak
                     /note="color: #a6acb3"
     promoter        6689..6793
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             6794..7654
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 6794 .. 6862 / #ccffcc / signal sequence
                        2: 6863 .. 7654 / #ccffcc
                     Cleavage site after base 6862"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7825..8413
                     /direction=RIGHT
                     /label=pUC ori
                     /note="/application_notes=Facilitates plasmid replication 
                     in E.coli; regulates high-copy plasmid number (500-700)."
                     /note="/description=pUC origin of replication"
                     /note="/full_name=pUC origin of replication"
                     /note="/official_designation=pUC ori"
                     /note="/uuid=64a8cb3def9f49deb6752795c2d893a8"
                     /note="color: #ce0084"
     protein_bind    8701..8722
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     promoter        8737..8767
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                        1: 8737 .. 8742 / #ffffff / -35
                        2: 8743 .. 8760 / #ffffff
                        3: 8761 .. 8767 / #ffffff / -10"
     protein_bind    8775..8791
                     /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)."
                     /note="color: #31849b"
     primer_bind     8799..8815
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     promoter        8836..8854
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        8836..8854
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
ORIGIN
        1 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
       61 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
      121 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
      181 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
      241 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
      301 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
      361 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
      421 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
      481 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
      541 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
      601 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
      661 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
      721 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
      781 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
      841 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
      901 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
      961 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     1021 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     1081 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     1141 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     1201 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     1261 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     1321 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     1381 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcta
     1801 gcttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     1861 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     1921 actagtgatt atcggatcaa ctttgtatag aaaagttgtc tagagggcag agcgcacatc
     1981 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     2041 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2101 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2161 tgccgccaga acacagggat cccaagtttg tacaaaaaag caggctgcca ccatgctcag
     2221 gctgctcttg gctctcaact tattcccttc aattcaagta acaggactcg aggaacaaaa
     2281 actcatctca gaagaggatc tggaacaaaa gctcatttca gaagaagatc tgctcgaggt
     2341 cgacggagac gccgacatcc agatgacaca gactacatcc tccctgtctg cctctctggg
     2401 agacagagtc accatcagtt gcagggcaag tcaggacatc tctaagtatt tgaattggta
     2461 tcagcagaaa ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc
     2521 aggagtccca tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag
     2581 caacctggag caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta
     2641 cacgttcgga ggggggacta agttggaaat aacaggctcc acctctggat ccggcaagcc
     2701 cggatctggc gagggatcca ccaagggcga ggtgaaactg caggagtcag gacctggcct
     2761 ggtggcgccc tcacagagcc tgtccgtcac atgcactgtc tcaggggtct cattacccga
     2821 ctatggtgta agctggattc gccagcctcc acgaaagggt ctggagtggc tgggagtaat
     2881 atggggtagt gaaaccacat actataattc agctctcaaa tccagactga ccatcatcaa
     2941 ggacaactcc aagagccaag ttttcttaaa aatgaacagt ctgcaaactg atgacacagc
     3001 catttactac tgtgccaaac attattacta cggtggtagc tatgctatgg actactgggg
     3061 tcaaggaacc tcagtcaccg tctccggtac cattgaagtt atgtatcctc ctccttacct
     3121 agacaatgag aagagcaatg gaaccattat ccatgtgaaa gggaaacacc tttgtccaag
     3181 tcccctattt cccggacctt ctaagccctt ttgggtgctg gtggtggttg gtggagtcct
     3241 ggcttgctat agcttgctag taacagtggc ctttattatt ttctgggtga ggaaacgggg
     3301 cagaaagaaa ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca
     3361 agaggaagat ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag
     3421 taagaggagc aggctcctgc acagtgacta catgaacatg actcccaggc ggcccggacc
     3481 cacccgcaag cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctcctc
     3541 cggactgaga gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa
     3601 ccagctctat aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag
     3661 gcgtggccgg gaccctgaga tggggggaaa gccgcagaga aggaagaacc ctcaggaagg
     3721 cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa
     3781 aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac
     3841 caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgctccg gacgtacggc
     3901 cacgaacttc tctctgttaa agcaagcagg cgacgtggaa gaaaaccccg gtcccgtgag
     3961 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     4021 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     4081 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     4141 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     4201 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     4261 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     4321 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     4381 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     4441 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     4501 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     4561 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     4621 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aagggccccg
     4681 tacccagctt tcttgtacaa agtggtgata atcgaattcc gataatcaac ctctggatta
     4741 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     4801 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     4861 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     4921 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     4981 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     5041 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     5101 cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg
     5161 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     5221 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     5281 gagtcggatc tccctttggg ccgcctcccc gcatcgggaa ttcccgcggt tcgctttaag
     5341 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     5401 ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct
     5461 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     5521 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     5581 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag
     5641 ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga
     5701 gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5761 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5821 atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact
     5881 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     5941 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     6001 ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     6061 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     6121 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     6181 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     6241 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     6301 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     6361 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     6421 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     6481 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     6541 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     6601 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     6661 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     6721 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     6781 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     6841 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     6901 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     6961 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     7021 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     7081 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     7141 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     7201 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     7261 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     7321 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     7381 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     7441 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     7501 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     7561 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     7621 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     7681 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     7741 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     7801 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     7861 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7921 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     7981 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     8041 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     8101 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     8161 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     8221 agaaagcgcc acgcttcccg aagagagaaa ggcggacagg tatccggtaa gcggcagggt
     8281 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     8341 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     8401 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     8461 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     8521 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     8581 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     8641 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     8701 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     8761 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     8821 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctt
//
