LOCUS pLentiRNACRISPR_006 - hU6-DR_BsmBI-EFS-RfxCas13d-NES-2A-Puro-WPRE 11728 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses RfxCas13d in mammalian cells, localized in the cytoplasm.
For cloning of guide RNAs compatible with RfxCas13d. Contains a 5'
direct repeat. Clone using BsmBI. F overhang aaac. R overhang aaaa.
ACCESSION .
VERSION .
KEYWORDS pLentiRNACRISPR_006 - hU6-DR_BsmBI-EFS-RfxCas13d-NES-2A-Puro-WPRE
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11728)
AUTHORS Wessels HH, Mendez-Mancilla A, Guo X, Legut M, Daniloski Z, Sanjana
NE
TITLE Massively parallel Cas13 screens reveal principles for guide RNA
design.
JOURNAL Nat Biotechnol. 2020 Jun;38(6):722-727. doi:
10.1038/s41587-020-0456-9. Epub 2020 Mar 16.
PUBMED 32518401
REFERENCE 2 (bases 1 to 11728)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11728
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(58..77)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 249..628
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 630..828
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 783..803
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 846..1026
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1073..1198
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1691..1924
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2109..2153
/codon_start=1
/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"
/translation="KNEQELLELDKWASL"
misc_feature 2451..2568
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2619..2859
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 2619..2639
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 2790..2809
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 2869..2898
/label=RfxCas13d DR30
/note="5' processed 30-base direct repeat for an RfxCas13d
gRNA (Konermann et al., 2018)"
promoter 2977..3188
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 3213..6110
/codon_start=1
/product="RNA-targeting Type VI CRISPR protein from
Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
2018)"
/label=RfxCas13d
/translation="MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS
IRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLE
KRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV
YTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYII
NYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITN
ELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRK
DMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFV
INLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPA
GRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAF
FKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDE
NGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ
KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRE
NAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLE
EKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKK
AEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVH
AYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCV
PFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGN"
CDS 6117..6143
/codon_start=1
/product="nuclear export signal from the HIV Rev protein
(Fischer et al., 1995)"
/label=NES
/translation="LPPLERLTL"
CDS 6162..6218
/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"
CDS 6219..6815
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind 6712..6732
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 6831..7419
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(6884..6904)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(7302..7313)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 7491..7724
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(7750..7767)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 7756..7980
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 8026..8454
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(8113..8132)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 8323..8344
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(8463..8483)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 8468..8797
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 8648..8783
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 8710..8729
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 8845..8892
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 8911..9285
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 9415..9536
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9452..9471)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9506..9525
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(9585..9601)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9585..9601)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(9598..9620)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9609..9625
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(9633..9663)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9678..9699
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(9816..9833)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(9987..10575)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(10067..10086)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10746..11606)
/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 11369..11388
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(11607..11711)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
61 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
121 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
181 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
241 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
301 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
361 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
421 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
481 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
541 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
601 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
661 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
721 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
781 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct
841 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
901 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
961 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
1021 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
1081 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
1141 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
1201 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa
1261 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt
1321 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc
1381 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta
1441 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga
1501 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag
1561 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
1621 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
1681 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
1741 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
1801 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
1861 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
1921 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
1981 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
2041 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
2101 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
2161 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
2221 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
2281 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag
2341 gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat
2401 cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga
2461 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
2521 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
2581 agggacagca gagatccagt ttggttaatt aaggtaccga gggcctattt cccatgattc
2641 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
2701 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
2761 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
2821 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgaa cccctaccaa
2881 ctggtcgggg tttgaaacgg agacggacgt ctcttttttt tgaattcgct agctaggtct
2941 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
3001 tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg
3061 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
3121 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
3181 gaacacagga ccggttctag agcgctgcca ccatgatcga aaaaaaaaag tccttcgcca
3241 agggcatggg cgtgaagtcc acactcgtgt ccggctccaa agtgtacatg acaaccttcg
3301 ccgaaggcag cgacgccagg ctggaaaaga tcgtggaggg cgacagcatc aggagcgtga
3361 atgagggcga ggccttcagc gctgaaatgg ccgataaaaa cgccggctat aagatcggca
3421 acgccaaatt cagccatcct aagggctacg ccgtggtggc taacaaccct ctgtatacag
3481 gacccgtcca gcaggatatg ctcggcctga aggaaactct ggaaaagagg tacttcggcg
3541 agagcgctga tggcaatgac aatatttgta tccaggtgat ccataacatc ctggacattg
3601 aaaaaatcct cgccgaatac attaccaacg ccgcctacgc cgtcaacaat atctccggcc
3661 tggataagga cattattgga ttcggcaagt tctccacagt gtatacctac gacgaattca
3721 aagaccccga gcaccatagg gccgctttca acaataacga taagctcatc aacgccatca
3781 aggcccagta tgacgagttc gacaacttcc tcgataaccc cagactcggc tatttcggcc
3841 aggccttttt cagcaaggag ggcagaaatt acatcatcaa ttacggcaac gaatgctatg
3901 acattctggc cctcctgagc ggactgaggc actgggtggt ccataacaac gaagaagagt
3961 ccaggatctc caggacctgg ctctacaacc tcgataagaa cctcgacaac gaatacatct
4021 ccaccctcaa ctacctctac gacaggatca ccaatgagct gaccaactcc ttctccaaga
4081 actccgccgc caacgtgaac tatattgccg aaactctggg aatcaaccct gccgaattcg
4141 ccgaacaata tttcagattc agcattatga aagagcagaa aaacctcgga ttcaatatca
4201 ccaagctcag ggaagtgatg ctggacagga aggatatgtc cgagatcagg aaaaatcata
4261 aggtgttcga ctccatcagg accaaggtct acaccatgat ggactttgtg atttataggt
4321 attacatcga agaggatgcc aaggtggctg ccgccaataa gtccctcccc gataatgaga
4381 agtccctgag cgagaaggat atctttgtga ttaacctgag gggctccttc aacgacgacc
4441 agaaggatgc cctctactac gatgaagcta atagaatttg gagaaagctc gaaaatatca
4501 tgcacaacat caaggaattt aggggaaaca agacaagaga gtataagaag aaggacgccc
4561 ctagactgcc cagaatcctg cccgctggcc gtgatgtttc cgccttcagc aaactcatgt
4621 atgccctgac catgttcctg gatggcaagg agatcaacga cctcctgacc accctgatta
4681 ataaattcga taacatccag agcttcctga aggtgatgcc tctcatcgga gtcaacgcta
4741 agttcgtgga ggaatacgcc tttttcaaag actccgccaa gatcgccgat gagctgaggc
4801 tgatcaagtc cttcgctaga atgggagaac ctattgccga tgccaggagg gccatgtata
4861 tcgacgccat ccgtatttta ggaaccaacc tgtcctatga tgagctcaag gccctcgccg
4921 acaccttttc cctggacgag aacggaaaca agctcaagaa aggcaagcac ggcatgagaa
4981 atttcattat taataacgtg atcagcaata aaaggttcca ctacctgatc agatacggtg
5041 atcctgccca cctccatgag atcgccaaaa acgaggccgt ggtgaagttc gtgctcggca
5101 ggatcgctga catccagaaa aaacagggcc agaacggcaa gaaccagatc gacaggtact
5161 acgaaacttg tatcggaaag gataagggca agagcgtgag cgaaaaggtg gacgctctca
5221 caaagatcat caccggaatg aactacgacc aattcgacaa gaaaaggagc gtcattgagg
5281 acaccggcag ggaaaacgcc gagagggaga agtttaaaaa gatcatcagc ctgtacctca
5341 ccgtgatcta ccacatcctc aagaatattg tcaatatcaa cgccaggtac gtcatcggat
5401 tccattgcgt cgagcgtgat gctcaactgt acaaggagaa aggctacgac atcaatctca
5461 agaaactgga agagaaggga ttcagctccg tcaccaagct ctgcgctggc attgatgaaa
5521 ctgcccccga taagagaaag gacgtggaaa aggagatggc tgaaagagcc aaggagagca
5581 ttgacagcct cgagagcgcc aaccccaagc tgtatgccaa ttacatcaaa tacagcgacg
5641 agaagaaagc cgaggagttc accaggcaga ttaacaggga gaaggccaaa accgccctga
5701 acgcctacct gaggaacacc aagtggaatg tgatcatcag ggaggacctc ctgagaattg
5761 acaacaagac atgtaccctg ttcagaaaca aggccgtcca cctggaagtg gccaggtatg
5821 tccacgccta tatcaacgac attgccgagg tcaattccta cttccaactg taccattaca
5881 tcatgcagag aattatcatg aatgagaggt acgagaaaag cagcggaaag gtgtccgagt
5941 acttcgacgc tgtgaatgac gagaagaagt acaacgatag gctcctgaaa ctgctgtgtg
6001 tgcctttcgg ctactgtatc cccaggttta agaacctgag catcgaggcc ctgttcgata
6061 ggaacgaggc cgccaagttc gacaaggaga aaaagaaggt gtccggcaat cttcaactgc
6121 ctccacttga aagactgaca ctgggtagtt ccggatccgg cgcaacaaac ttctctctgc
6181 tgaaacaagc cggagatgtc gaagagaatc ctggaccgac cgagtacaag cccacggtgc
6241 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
6301 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
6361 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
6421 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
6481 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
6541 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
6601 gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
6661 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
6721 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
6781 gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc
6841 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
6901 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
6961 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
7021 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
7081 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
7141 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
7201 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
7261 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
7321 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
7381 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
7441 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
7501 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
7561 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
7621 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
7681 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc
7741 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
7801 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
7861 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
7921 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
7981 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
8041 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
8101 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
8161 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
8221 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
8281 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
8341 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
8401 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
8461 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
8521 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
8581 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
8641 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
8701 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
8761 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
8821 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
8881 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
8941 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
9001 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
9061 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
9121 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
9181 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
9241 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat
9301 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
9361 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
9421 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
9481 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
9541 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
9601 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
9661 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
9721 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
9781 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
9841 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
9901 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
9961 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
10021 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
10081 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
10141 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
10201 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
10261 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
10321 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
10381 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
10441 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
10501 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
10561 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
10621 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
10681 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
10741 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
10801 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
10861 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
10921 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
10981 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
11041 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
11101 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
11161 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
11221 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
11281 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
11341 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
11401 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
11461 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
11521 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
11581 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
11641 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
11701 ggggttccgc gcacatttcc ccgaaaag
//
