pAsRed2-C1载体描述
pAsRed2-C1 encodes AsRed2, a variant of Anemonia sulcata red fluorescent protein (1, 2). AsRed2 has been engineered for brighter fluorescence (Clontech, unpublished data). The AsRed2 coding sequence also contains a series of silent base-pair changes, which correspond to human codon-usage preferences for optimal expression in mammalian cells (3). Additionally, an upstream sequence—located just 5' to the AsRed2 start codon—has been converted to a Kozak consensus translation initiation site (4) to further increase the translation efficiency in eukaryotic cells. The multiple cloning site (MCS) in pAsRed2-C1 is positioned between the AsRed2 coding sequence and a pair of SV40 polyadenylation signals (SV40 poly A). Thus, genes cloned into the MCS will be expressed as fusions to the C-terminus of AsRed2 if they are in the same reading frame as AsRed2 and there are no intervening stop codons. Expression of AsRed2 is driven by the cytomegalovirus immediate-early promoter (PCMV IE). The SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of AsRed2 mRNA.
The vector backbone contains an SV40 origin (SV40 ori) for replication in mammalian cells that express the SV40 T-antigen, a pUC origin of replication (pUC ori) for propagation in E. coli, and an f1 origin (f1 ori) for single-stranded DNA production. A neomycin resistance cassette—consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK poly A) gene—allows stably transfected eukaryotic cells to be selected using G418 (5). A bacterial promoter (P) upstream of this cassette drives expression of the Neor/Kanr gene in E. coli hosts, which can be selected with kanamycin.
Fusions to the C terminus of AsRed2 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo (AsRed2 excitation maximum = 576 nm; AsRed2 emission maximum = 592 nm). The target gene should be cloned into pAsRed2-C1 so that it is in frame with the AsRed2 coding sequence, with no intervening, in-frame stop codons. The recombinant pAsRed2-C1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (available from Clontech; Cat. Nos. 631307 & 631308). We recommend selecting mammalian cell cultures in 500–1,300 μg/ml G418, depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of G418 to determine the optimal selection concentration. Unmodified (i.e., non-recombinant) pAsRed2-C1 can also be used simply to express AsRed2 in cells of interest (e.g., as a transfection marker).
Propagation in E. coli
Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM109 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
E. coli replication origin: pUC
Copy number: ~500
Plasmid incompatibility group: pMB1/Col E1
LOCUS pAsRed2-C1 4710 bp DNA SYN
DEFINITION pAsRed2-C1
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..4710
/organism="pAsRed2-C1"
/mol_type="other DNA"
promoter 10..562
/label="CMV_immearly_promoter"
misc_feature 65..352
/label="CAG_enhancer"
misc_feature 519..539
/label="CMV_fwd_primer"
promoter 520..589
/label="CMV_promoter"
CDS complement(598..1407)
/label="ORF frame 1"
/translation="MADYDQLSRSGGSRARGTVDCRIRSLSSRSESGVVAQLGGGGVA
VAAHGGLVLLVALALLHLLQDLDAVVLEVEAGHLQGGGGLLGAVGGVQVAGQVATAGA
LQGHQGLAAQHAVHLVHDLGGGLPAAGLVLHHGAVGGEVVAQDLHLVHQAVAVQGGVL
VGGQEAAVLVGGGALPGEALGEGLLEVVGDAGHVLDEGLGAVHAGGGQDVEGEGQGAA
LDHLDLHLLGALQGVALALAGALEVVAVHGALDGGPEGHGLLQQRGHGGDR*"
CDS 613..1389
/label="ORF frame 1"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
gene 616..1386
/label="AsRed2"
/gene="AsRed2"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
terminator 1391..1619
/label="SV40_PA_terminator"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
misc_feature 1589..1608
/label="EBV_rev_primer"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
rep_origin complement(1758..2064)
/label="f1_origin"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
promoter 2143..2171
/label="AmpR_promoter"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
misc_feature complement(2237..2257)
/label="pBABE_3_primer"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
misc_feature complement(2243..2458)
/label="SV40_enhancer"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
promoter 2255..2523
/label="SV40_promoter"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
rep_origin 2422..2499
/label="SV40_origin"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
misc_feature 2484..2503
/label="SV40pro_F_primer"
/translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIE
VIEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFL
TAHQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLM
ALECPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAV
GRYCDAAPSKLGHNSGLRSRAQASNSAVDGTAGPGSTGSR*"
CDS 2606..3400
/label="ORF frame 2"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
gene 2609..3397
/label="NeoR/KanR"
/gene="NeoR/KanR"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
CDS complement(2915..3451)
/label="ORF frame 3"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
terminator 3575..3844
/label="TK_PA_terminator"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
rep_origin 3992..4611
/label="pBR322_origin"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
ORIGIN
1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG
61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT
121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA
181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC
241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA
301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC
361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG
421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG
481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT
541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA
601 CCGGTCGCCA CCATGGCCTC TTTGCTGAAG AAGACCATGC CCTTCAGGAC CACCATCGAG
661 GGCACCGTGA ACGGCCACTA CTTCAAGTGC ACCGGCAAGG GCGAGGGCAA CCCCCTGGAG
721 GGCACCCAGG AGATGAAGAT CGAGGTGATC GAGGGCGGCC CCCTGCCCTT CGCCTTCCAC
781 ATCCTGTCCA CCTCCTGCAT GTACGGCTCC AAGGCCTTCA TCAAGTACGT GTCCGGCATC
841 CCCGACTACT TCAAGCAGTC CCTCCCCGAG GGCTTCACCT GGGAGCGCAC CACCACCTAC
901 GAGGACGGCG GCTTCCTGAC CGCCCACCAG GACACCTCCC TGGACGGCGA CTGCCTGGTG
961 TACAAGGTGA AGATCCTGGG CAACAACTTC CCCGCCGACG GCCCCGTGAT GCAGAACAAG
1021 GCCGGCCGCT GGGAGCCCTC CACCGAGATC GTGTACGAGG TGGACGGCGT GCTGCGCGGC
1081 CAGTCCCTGA TGGCCCTGGA GTGCCCCGGC GGTCGCCACC TGACCTGCCA CCTGCACACC
1141 ACCTACCGCT CCAAGAAGCC CGCCTCCGCC CTGAAGATGC CCGGCTTCCA CTTCGAGGAC
1201 CACCGCATCG AGATCCTGGA GGAGGTGGAG AAGGGCAAGT GCTACAAGCA GTACGAGGCC
1261 GCCGTGGGCC GCTACTGCGA CGCCGCCCCC TCCAAGCTGG GCCACAACTC CGGACTCAGA
1321 TCTCGAGCTC AAGCTTCGAA TTCTGCAGTC GACGGTACCG CGGGCCCGGG ATCCACCGGA
1381 TCTAGATAAC TGATCATAAT CAGCCATACC ACATTTGTAG AGGTTTTACT TGCTTTAAAA
1441 AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA ATGCAATTGT TGTTGTTAAC
1501 TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT
1561 AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAA
1621 CGCGTAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG
1681 CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC
1741 CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA
1801 CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC
1861 ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG
1921 GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA
1981 GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC
2041 CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG
2101 AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT
2161 CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA AAAAGGAAGA GTCCTGAGGC
2221 GGAAAGAACC AGCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA
2281 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC
2341 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA
2401 GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG
2461 CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG
2521 CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AGATCGATCA
2581 AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC
2641 GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTC
2701 TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGA
2761 CCTGTCCGGT GCCCTGAATG AACTGCAAGA CGAGGCAGCG CGGCTATCGT GGCTGGCCAC
2821 GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT
2881 GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA
2941 AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCC
3001 ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCT
3061 TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGC
3121 CAGGCTCAAG GCGAGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG
3181 CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT
3241 GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCT
3301 TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCA
3361 GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAA
3421 ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC
3481 TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC
3541 GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCTAGGGGGA GGCTAACTGA AACACGGAAG
3601 GAGACAATAC CGGAAGGAAC CCGCGCTATG ACGGCAATAA AAAGACAGAA TAAAACGCAC
3661 GGTGTTGGGT CGTTTGTTCA TAAACGCGGG GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT
3721 ACCCCACCGA GACCCCATTG GGGCCAATAC GCCCGCGTTT CTTCCTTTTC CCCACCCCAC
3781 CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC AGGCCCTGCC
3841 ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA
3901 AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT
3961 TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT
4021 TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT
4081 TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG
4141 ATACCAAATA CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA
4201 GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT
4261 AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG
4321 GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG
4381 AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC
4441 AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA
4501 AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT
4561 TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA
4621 CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT
4681 TCTGTGGATA ACCGTATTAC CGCCATGCAT
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