质粒名称: | pCW57-MCS1-P2A-MCS2 (RFP) |
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出品公司: | Addgene |
目录编号: | 80923 |
存储实验室: | Adam Karpf |
载体骨架基本信息 |
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载体名称: | pCW57.1 |
载体出品公司: | Broad Insitute |
载体抗性: | 氨苄青霉素 |
空载体大小: | 7834 bp |
完整质粒大小: | -- |
载体修饰: | 用turbo-RFP代替嘌呤霉素对pCW57-MCS1-2A-MCS2进行了改性。 |
载体类型: | 哺乳动物表达,慢病毒;强力霉素诱导;P2A自切割肽;Turbo RFP reporter |
筛选标记: | RFP |
高拷贝/低拷贝: | -- |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | -- |
物种来源: | -- |
插入大小: | -- |
基因突变: | -- |
GenBank编号 | -- |
启动子 | TRE |
标签/融合蛋白: | -- |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | pCW57.MCS_Seq_F CGTATGTCGAGGTAGGCGTG |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pCW57-MCS1-P2A-MCS2 (RFP) was a gift from Adam Karpf (Addgene plasmid # 80923 ; http://n2t.net/addgene:80923 ; RRID:Addgene_80923) |
参考文献部分: | Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression in Cancer. Barger CJ, Branick C, Chee L, Karpf AR. Cancers (Basel). 2019 Feb 21;11(2). pii: cancers11020251. doi: 10.3390/cancers11020251. 10.3390/cancers11020251 PubMed 30795624 |
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载体序列
LOCUS pCW57-MCS1-P2A-MCS2 (RFP) 7834 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION All-in-one doxycycline inducible lentiviral vector for expression of one or two genes using the P2A self-cleaving peptide.. ACCESSION . VERSION . KEYWORDS pCW57-MCS1-P2A-MCS2 (RFP) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7834) TITLE All-in-one doxycycline inducible lentiviral vector for expression of one or two genes. JOURNAL Unpublished REFERENCE 2 (bases 1 to 7834) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7834 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 41..158 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 212..526 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 220..238 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 256..274 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 291..309 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 327..345 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 363..381 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 398..416 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 434..452 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 499..523 /label=LNCX /note="Human CMV promoter, forward primer" CDS 567..623 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" promoter 651..1161 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 1080..1098 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 1185..1877 /codon_start=1 /product="red fluorescent protein from Entacmaea quadricolor" /label=TurboRFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC DLPSKLGHR" CDS 1893..1946 /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 1947..2693 /codon_start=1 /product="improved tetracycline-controlled transactivator" /label=rtTA-Advanced /note="In the Tet-On(R) system, rtTA-Advanced binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline." /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" primer_bind complement(2733..2753) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3151..3162) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3367..3600 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3672..3793 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3709..3728) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3763..3782 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3833..3968 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3895..3914 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 3968..4039 /gene="bla" /label=AmpR promoter CDS 4040..4900 /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 complement(4258..4277) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5071..5659 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5560..5579 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5813..5830 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5947..5968 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5983..6013 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6021..6037 /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 6026..6048 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6045..6061 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6045..6061 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6080..6100 /label=T3 /note="T3 promoter, forward primer" promoter 6082..6100 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6128..6354 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6355..6535 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6582..6707 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7200..7433 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." ORIGIN 1 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga 61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta 121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca 181 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac 241 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag 301 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt 361 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt 421 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg 481 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta 541 gcgaattcgt cgacaccggt tccggagcca cgaacttctc tctgttaaag caagcaggag 601 acgtggaaga aaaccccggt cctaggctgc agacgcgttg tacaggatcc ggggttgggg 661 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt 721 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc 781 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc 841 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca 901 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg 961 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa 1021 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg 1081 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga 1141 atcaccgacc tctctcccca gcaattcacc cgcgcgcggc caccatgagc gagctgatca 1201 aggagaacat gcacatgaag ctgtacatgg agggcaccgt gaacaaccac cacttcaagt 1261 gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaag atcaaggtgg 1321 tcgagggcgg ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca 1381 gcaaagcctt catcaaccac acccagggca tccccgactt ctttaagcag tccttccctg 1441 agggcttcac atgggagaga atcaccacat acgaagacgg gggcgtgctg accgctaccc 1501 aggacaccag cttccagaac ggctgcatca tctacaacgt caagatcaac ggggtgaact 1561 tcccatccaa cggccctgtg atgcagaaga aaacacgcgg ctgggaggcc aacaccgaga 1621 tgctgtaccc cgctgacggc ggcctgagag gccacagcca gatggccctg aagctcgtgg 1681 gcgggggcta cctgcactgc tccttcaaga ccacatacag atccaagaaa cccgctaaga 1741 acctcaagat gcccggcttc cacttcgtgg accacagact ggaaagaatc aaggaggccg 1801 acaaagagac ctacgtcgag cagcacgaga tggctgtggc caagtactgc gacctcccta 1861 gcaaactggg gcacagattc gaaggaagcg gagaaggtag aggttctctc ctcacttgtg 1921 gtgatgttga agaaaaccct ggtccaatgt ctagactgga caagagcaaa gtcataaacg 1981 gagctctgga attactcaat ggtgtcggta tcgaaggcct gacgacaagg aaactcgctc 2041 aaaagctggg agttgagcag cctaccctgt actggcacgt gaagaacaag cgggccctgc 2101 tcgatgccct gccaatcgag atgctggaca ggcatcatac ccacttctgc cccctggaag 2161 gcgagtcatg gcaagacttt ctgcggaaca acgccaagtc ataccgctgt gctctcctct 2221 cacatcgcga cggggctaaa gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa 2281 ccctggaaaa tcagctcgcg ttcctgtgtc agcaaggctt ctccctggag aacgcactgt 2341 acgctctgtc cgccgtgggc cactttacac tgggctgcgt attggaggaa caggagcatc 2401 aagtagcaaa agaggaaaga gagacaccta ccaccgattc tatgccccca cttctgagac 2461 aagcaattga gctgttcgac cggcagggag ccgaacctgc cttccttttc ggcctggaac 2521 taatcatatg tggcctggag aaacagctaa agtgcgaaag cggcgggccg accgacgccc 2581 ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata 2641 tgctgcctgc tgacgctctt gacgattttg accttgacat gctccccggg taactaagta 2701 aggatcgatc caagatatcg tattcttaac tatgttgctc cttttacgct atgtggatac 2761 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc 2821 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt 2881 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc 2941 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc 3001 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg 3061 gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt 3121 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc 3181 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt 3241 cggatctccc tttgggccgc ctccccgcct gtttcgcctc ggcgtccgga ctagaggtac 3301 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg 3361 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg 3421 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 3481 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 3541 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 3601 gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag 3661 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3721 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3781 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc 3841 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 3901 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt 3961 tggaggcctt tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 4021 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 4081 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 4141 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 4201 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 4261 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 4321 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 4381 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 4441 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 4501 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 4561 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 4621 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 4681 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 4741 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 4801 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 4861 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 4921 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 4981 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 5041 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 5101 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 5161 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct 5221 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 5281 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 5341 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 5401 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 5461 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 5521 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 5581 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 5641 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 5701 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 5761 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 5821 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc 5881 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 5941 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc 6001 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga 6061 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca 6121 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag 6181 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg 6241 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact 6301 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct 6361 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 6421 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 6481 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 6541 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 6601 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 6661 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 6721 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 6781 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 6841 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 6901 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 6961 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 7021 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 7081 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 7141 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 7201 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 7261 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 7321 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 7381 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 7441 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 7501 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 7561 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 7621 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 7681 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 7741 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 7801 tcgtttcaga cccacctccc aaccccgagg ggac //
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