LOCUS       sequence-466772-       12987 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses RfxCas13d in mammalian cells for knockdown of target RNAs 
            in combination with compatible crRNAs. Localized to the nucleus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12987)
  AUTHORS   Hart SK, Wessels HH, Mendez-Mancilla A, Muller S, Drabavicius G, 
            Choi O, Sanjana NE
  TITLE     Low copy CRISPR-Cas13d mitigates collateral RNA cleavage.
  JOURNAL   bioRxiv [Preprint]. 2024 May 14:2024.05.13.594039. doi: 
            10.1101/2024.05.13.594039.
  PUBMED    38798586
REFERENCE   2  (bases 1 to 12987)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 May 14:2024.05.13.594039. doi: 
            10.1101/2024.05.13.594039."
COMMENT     Sequence Label: 
            pLentiRNACRISPR_009-TetO-NLS-RfxCas13d-NLS-WPRE-EFS-rtTA3-2A-EGFP
FEATURES             Location/Qualifiers
     source          1..12987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /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    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2365..2385
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    2430..2448
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2465..2483
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2501..2519
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2537..2555
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2572..2590
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2608..2626
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        2638..2676
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2673..2697
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2741..2761
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2771..5668
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             5678..5698
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5708..5734
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    5784..6372
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5837..5857)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6255..6266)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6375..6391)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6376..6392)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        6437..6648
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             6673..7377
                     /codon_start=1
                     /product="reverse tetracycline transactivator 3"
                     /label=rtTA3
                     /note="activates expression from Tet-responsive promoters 
                     in the presence of doxycycline"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPG"
     CDS             7387..7443
                     /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             join(7447..7449,7450..8163)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(7489..7510)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7750..7769)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8097..8118
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             8555..8735
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(8761..8778)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8767..8991
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9037..9465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9124..9143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9334..9355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9474..9494)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9479..9808
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9659..9794
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9721..9740
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9856..9903
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9922..10296
                     /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    10426..10547
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10463..10482)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10517..10536
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10596..10612)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10596..10612)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10609..10631)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10620..10636
                     /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(join(10644..10650,10651..10668,10669..10674))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10689..10710
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10827..10844)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10998..11586)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11078..11097)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11757..12548,12549..12617))
                     /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     12380..12399
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12618..12722)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12797..12816)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttaacaactt tgtatagaaa agttggctcc gaattcgccc
     2401 ttcaggtccg aggttctaga cgagtttact ccctatcagt gatagagaac gatgtcgagt
     2461 ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa
     2521 cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt atccctatca
     2581 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgaggt
     2641 aggcgtgtac ggtgggaggc ctatataagc agagctcgtt tagtgaaccg tcagatcgca
     2701 aagggcgaat tcgacccaag tttgtacagc caccatgagc cccaagaaga agagaaaggt
     2761 ggaggccagc atcgaaaaaa aaaagtcctt cgccaagggc atgggcgtga agtccacact
     2821 cgtgtccggc tccaaagtgt acatgacaac cttcgccgaa ggcagcgacg ccaggctgga
     2881 aaagatcgtg gagggcgaca gcatcaggag cgtgaatgag ggcgaggcct tcagcgctga
     2941 aatggccgat aaaaacgccg gctataagat cggcaacgcc aaattcagcc atcctaaggg
     3001 ctacgccgtg gtggctaaca accctctgta tacaggaccc gtccagcagg atatgctcgg
     3061 cctgaaggaa actctggaaa agaggtactt cggcgagagc gctgatggca atgacaatat
     3121 ttgtatccag gtgatccata acatcctgga cattgaaaaa atcctcgccg aatacattac
     3181 caacgccgcc tacgccgtca acaatatctc cggcctggat aaggacatta ttggattcgg
     3241 caagttctcc acagtgtata cctacgacga attcaaagac cccgagcacc atagggccgc
     3301 tttcaacaat aacgataagc tcatcaacgc catcaaggcc cagtatgacg agttcgacaa
     3361 cttcctcgat aaccccagac tcggctattt cggccaggcc tttttcagca aggagggcag
     3421 aaattacatc atcaattacg gcaacgaatg ctatgacatt ctggccctcc tgagcggact
     3481 gaggcactgg gtggtccata acaacgaaga agagtccagg atctccagga cctggctcta
     3541 caacctcgat aagaacctcg acaacgaata catctccacc ctcaactacc tctacgacag
     3601 gatcaccaat gagctgacca actccttctc caagaactcc gccgccaacg tgaactatat
     3661 tgccgaaact ctgggaatca accctgccga attcgccgaa caatatttca gattcagcat
     3721 tatgaaagag cagaaaaacc tcggattcaa tatcaccaag ctcagggaag tgatgctgga
     3781 caggaaggat atgtccgaga tcaggaaaaa tcataaggtg ttcgactcca tcaggaccaa
     3841 ggtctacacc atgatggact ttgtgattta taggtattac atcgaagagg atgccaaggt
     3901 ggctgccgcc aataagtccc tccccgataa tgagaagtcc ctgagcgaga aggatatctt
     3961 tgtgattaac ctgaggggct ccttcaacga cgaccagaag gatgccctct actacgatga
     4021 agctaataga atttggagaa agctcgaaaa tatcatgcac aacatcaagg aatttagggg
     4081 aaacaagaca agagagtata agaagaagga cgcccctaga ctgcccagaa tcctgcccgc
     4141 tggccgtgat gtttccgcct tcagcaaact catgtatgcc ctgaccatgt tcctggatgg
     4201 caaggagatc aacgacctcc tgaccaccct gattaataaa ttcgataaca tccagagctt
     4261 cctgaaggtg atgcctctca tcggagtcaa cgctaagttc gtggaggaat acgccttttt
     4321 caaagactcc gccaagatcg ccgatgagct gaggctgatc aagtccttcg ctagaatggg
     4381 agaacctatt gccgatgcca ggagggccat gtatatcgac gccatccgta ttttaggaac
     4441 caacctgtcc tatgatgagc tcaaggccct cgccgacacc ttttccctgg acgagaacgg
     4501 aaacaagctc aagaaaggca agcacggcat gagaaatttc attattaata acgtgatcag
     4561 caataaaagg ttccactacc tgatcagata cggtgatcct gcccacctcc atgagatcgc
     4621 caaaaacgag gccgtggtga agttcgtgct cggcaggatc gctgacatcc agaaaaaaca
     4681 gggccagaac ggcaagaacc agatcgacag gtactacgaa acttgtatcg gaaaggataa
     4741 gggcaagagc gtgagcgaaa aggtggacgc tctcacaaag atcatcaccg gaatgaacta
     4801 cgaccaattc gacaagaaaa ggagcgtcat tgaggacacc ggcagggaaa acgccgagag
     4861 ggagaagttt aaaaagatca tcagcctgta cctcaccgtg atctaccaca tcctcaagaa
     4921 tattgtcaat atcaacgcca ggtacgtcat cggattccat tgcgtcgagc gtgatgctca
     4981 actgtacaag gagaaaggct acgacatcaa tctcaagaaa ctggaagaga agggattcag
     5041 ctccgtcacc aagctctgcg ctggcattga tgaaactgcc cccgataaga gaaaggacgt
     5101 ggaaaaggag atggctgaaa gagccaagga gagcattgac agcctcgaga gcgccaaccc
     5161 caagctgtat gccaattaca tcaaatacag cgacgagaag aaagccgagg agttcaccag
     5221 gcagattaac agggagaagg ccaaaaccgc cctgaacgcc tacctgagga acaccaagtg
     5281 gaatgtgatc atcagggagg acctcctgag aattgacaac aagacatgta ccctgttcag
     5341 aaacaaggcc gtccacctgg aagtggccag gtatgtccac gcctatatca acgacattgc
     5401 cgaggtcaat tcctacttcc aactgtacca ttacatcatg cagagaatta tcatgaatga
     5461 gaggtacgag aaaagcagcg gaaaggtgtc cgagtacttc gacgctgtga atgacgagaa
     5521 gaagtacaac gataggctcc tgaaactgct gtgtgtgcct ttcggctact gtatccccag
     5581 gtttaagaac ctgagcatcg aggccctgtt cgataggaac gaggccgcca agttcgacaa
     5641 ggagaaaaag aaggtgtccg gcaattccgg atccggacct aagaaaaaga ggaaggtggc
     5701 ggccgcttac ccatacgatg ttccagatta cgcttaaggt acaaagtggt gaattccggc
     5761 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     5821 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     5881 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     5941 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     6001 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     6061 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     6121 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     6181 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     6241 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     6301 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     6361 ccgcctcccc gcatcgatac cgtcgacctc gagtaggtct tgaaaggagt gggaattggc
     6421 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     6481 ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     6541 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     6601 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga ccggttctag
     6661 agcgctgcca ccatgtctag gctggacaag agcaaagtca taaacggagc tctggaatta
     6721 ctcaatggtg tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt
     6781 gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga tgccctgcca
     6841 atcgagatgc tggacaggca tcatacccac ttctgccccc tggaaggcga gtcatggcaa
     6901 gactttctgc ggaacaacgc caagtcatac cgctgtgctc tcctctcaca tcgcgacggg
     6961 gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag
     7021 ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc tctgtccgcc
     7081 gtgggccact ttacactggg ctgcgtattg gaggaacagg agcatcaagt agcaaaagag
     7141 gaaagagaga cacctaccac cgattctatg cccccacttc tgagacaagc aattgagctg
     7201 ttcgaccggc agggagccga acctgccttc cttttcggcc tggaactaat catatgtggc
     7261 ctggagaaac agctaaagtg cgaaagcggc gggccgaccg acgcccttga cgattttgac
     7321 ttagacatgc tcccagccga tgcccttgac gattttgacc ttgacatgct ccccggggga
     7381 tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga gaatcctgga
     7441 ccgggagtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     7501 gacggcgacg taaacggcca caagttcagc gtgtctggcg agggcgaggg cgatgccacc
     7561 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     7621 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     7681 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     7741 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     7801 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     7861 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     7921 aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     7981 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     8041 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     8101 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     8161 taaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     8221 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct
     8281 gtggatctac cacacacaag gctacttccc tgattggcag aactacacac cagggccagg
     8341 gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa
     8401 ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg
     8461 gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca
     8521 tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag accagatctg
     8581 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     8641 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     8701 cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag
     8761 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     8821 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     8881 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     8941 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     9001 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     9061 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     9121 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     9181 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     9241 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     9301 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     9361 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     9421 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     9481 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     9541 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     9601 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     9661 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     9721 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     9781 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     9841 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     9901 gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg
     9961 cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc
    10021 gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc
    10081 caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag
    10141 ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc
    10201 atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc
    10261 aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc
    10321 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
    10381 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
    10441 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
    10501 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
    10561 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
    10621 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
    10681 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
    10741 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
    10801 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
    10861 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
    10921 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
    10981 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
    11041 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
    11101 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
    11161 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
    11221 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
    11281 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
    11341 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
    11401 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
    11461 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
    11521 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
    11581 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
    11641 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
    11701 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
    11761 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
    11821 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
    11881 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
    11941 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
    12001 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
    12061 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
    12121 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
    12181 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
    12241 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
    12301 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
    12361 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
    12421 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
    12481 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
    12541 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
    12601 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
    12661 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
    12721 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
    12781 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
    12841 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
    12901 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
    12961 gcgatgtacg ggccagatat acgcgtt
//
