LOCUS       sequence-457188-       13440 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 13440)
  TITLE     Low copy CRISPR-Cas13d mitigates collateral RNA cleavage
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13440)
  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: 
            "Unpublished"
COMMENT     Sequence Label: 
            pLentiRNACRISPR_010-TetO-NLS-RfxCas13d-2A-mCherry-NLS-WPRE-EFS-rtTA3
            -2A-Blast
FEATURES             Location/Qualifiers
     source          1..13440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..21
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    66..84
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    101..119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    137..155
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    173..191
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    208..226
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    244..262
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        274..312
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     309..333
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             377..397
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             407..3304
                     /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             3314..3334
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3344..3370
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3380..3436
                     /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             3437..4147
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3582..3600)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3850..3869
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    4194..4782
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4247..4267)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4665..4676)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4785..4801)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4786..4802)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        4847..5058
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             5083..5787
                     /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             5797..5853
                     /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             5854..6252
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             6644..6824
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6850..6867)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6856..7080
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7126..7554
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7213..7232)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7423..7444
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7563..7583)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7568..7897
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7748..7883
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7810..7829
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7945..7992
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8011..8385
                     /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    8515..8636
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8552..8571)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8606..8625
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8685..8701)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8685..8701)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8698..8720)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8709..8725
                     /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(8733..8739,8740..8757,8758..8763))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8778..8799
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8916..8933)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9087..9675)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9167..9186)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9846..10637,10638..10706))
                     /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     10469..10488
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10707..10811)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10886..10905)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11077..11456
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11458..11656
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             11674..11854
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11901..12026
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12519..12752
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12937..12981
                     /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    13279..13396
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 caactttgta tagaaaagtt ggctccgaat tcgcccttca ggtccgaggt tctagacgag
       61 tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag tgatagagaa
      121 cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc
      181 agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt atgtcgagtt
      241 tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat
      301 ataagcagag ctcgtttagt gaaccgtcag atcgcaaagg gcgaattcga cccaagtttg
      361 tacagccacc atgagcccca agaagaagag aaaggtggag gccagcatcg aaaaaaaaaa
      421 gtccttcgcc aagggcatgg gcgtgaagtc cacactcgtg tccggctcca aagtgtacat
      481 gacaaccttc gccgaaggca gcgacgccag gctggaaaag atcgtggagg gcgacagcat
      541 caggagcgtg aatgagggcg aggccttcag cgctgaaatg gccgataaaa acgccggcta
      601 taagatcggc aacgccaaat tcagccatcc taagggctac gccgtggtgg ctaacaaccc
      661 tctgtataca ggacccgtcc agcaggatat gctcggcctg aaggaaactc tggaaaagag
      721 gtacttcggc gagagcgctg atggcaatga caatatttgt atccaggtga tccataacat
      781 cctggacatt gaaaaaatcc tcgccgaata cattaccaac gccgcctacg ccgtcaacaa
      841 tatctccggc ctggataagg acattattgg attcggcaag ttctccacag tgtataccta
      901 cgacgaattc aaagaccccg agcaccatag ggccgctttc aacaataacg ataagctcat
      961 caacgccatc aaggcccagt atgacgagtt cgacaacttc ctcgataacc ccagactcgg
     1021 ctatttcggc caggcctttt tcagcaagga gggcagaaat tacatcatca attacggcaa
     1081 cgaatgctat gacattctgg ccctcctgag cggactgagg cactgggtgg tccataacaa
     1141 cgaagaagag tccaggatct ccaggacctg gctctacaac ctcgataaga acctcgacaa
     1201 cgaatacatc tccaccctca actacctcta cgacaggatc accaatgagc tgaccaactc
     1261 cttctccaag aactccgccg ccaacgtgaa ctatattgcc gaaactctgg gaatcaaccc
     1321 tgccgaattc gccgaacaat atttcagatt cagcattatg aaagagcaga aaaacctcgg
     1381 attcaatatc accaagctca gggaagtgat gctggacagg aaggatatgt ccgagatcag
     1441 gaaaaatcat aaggtgttcg actccatcag gaccaaggtc tacaccatga tggactttgt
     1501 gatttatagg tattacatcg aagaggatgc caaggtggct gccgccaata agtccctccc
     1561 cgataatgag aagtccctga gcgagaagga tatctttgtg attaacctga ggggctcctt
     1621 caacgacgac cagaaggatg ccctctacta cgatgaagct aatagaattt ggagaaagct
     1681 cgaaaatatc atgcacaaca tcaaggaatt taggggaaac aagacaagag agtataagaa
     1741 gaaggacgcc cctagactgc ccagaatcct gcccgctggc cgtgatgttt ccgccttcag
     1801 caaactcatg tatgccctga ccatgttcct ggatggcaag gagatcaacg acctcctgac
     1861 caccctgatt aataaattcg ataacatcca gagcttcctg aaggtgatgc ctctcatcgg
     1921 agtcaacgct aagttcgtgg aggaatacgc ctttttcaaa gactccgcca agatcgccga
     1981 tgagctgagg ctgatcaagt ccttcgctag aatgggagaa cctattgccg atgccaggag
     2041 ggccatgtat atcgacgcca tccgtatttt aggaaccaac ctgtcctatg atgagctcaa
     2101 ggccctcgcc gacacctttt ccctggacga gaacggaaac aagctcaaga aaggcaagca
     2161 cggcatgaga aatttcatta ttaataacgt gatcagcaat aaaaggttcc actacctgat
     2221 cagatacggt gatcctgccc acctccatga gatcgccaaa aacgaggccg tggtgaagtt
     2281 cgtgctcggc aggatcgctg acatccagaa aaaacagggc cagaacggca agaaccagat
     2341 cgacaggtac tacgaaactt gtatcggaaa ggataagggc aagagcgtga gcgaaaaggt
     2401 ggacgctctc acaaagatca tcaccggaat gaactacgac caattcgaca agaaaaggag
     2461 cgtcattgag gacaccggca gggaaaacgc cgagagggag aagtttaaaa agatcatcag
     2521 cctgtacctc accgtgatct accacatcct caagaatatt gtcaatatca acgccaggta
     2581 cgtcatcgga ttccattgcg tcgagcgtga tgctcaactg tacaaggaga aaggctacga
     2641 catcaatctc aagaaactgg aagagaaggg attcagctcc gtcaccaagc tctgcgctgg
     2701 cattgatgaa actgcccccg ataagagaaa ggacgtggaa aaggagatgg ctgaaagagc
     2761 caaggagagc attgacagcc tcgagagcgc caaccccaag ctgtatgcca attacatcaa
     2821 atacagcgac gagaagaaag ccgaggagtt caccaggcag attaacaggg agaaggccaa
     2881 aaccgccctg aacgcctacc tgaggaacac caagtggaat gtgatcatca gggaggacct
     2941 cctgagaatt gacaacaaga catgtaccct gttcagaaac aaggccgtcc acctggaagt
     3001 ggccaggtat gtccacgcct atatcaacga cattgccgag gtcaattcct acttccaact
     3061 gtaccattac atcatgcaga gaattatcat gaatgagagg tacgagaaaa gcagcggaaa
     3121 ggtgtccgag tacttcgacg ctgtgaatga cgagaagaag tacaacgata ggctcctgaa
     3181 actgctgtgt gtgcctttcg gctactgtat ccccaggttt aagaacctga gcatcgaggc
     3241 cctgttcgat aggaacgagg ccgccaagtt cgacaaggag aaaaagaagg tgtccggcaa
     3301 ttccggatcc ggacctaaga aaaagaggaa ggtggcggcc gcttacccat acgatgttcc
     3361 agattacgct ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga
     3421 ggagaaccct ggacctatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
     3481 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     3541 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     3601 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     3661 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     3721 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     3781 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     3841 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     3901 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     3961 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     4021 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     4081 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     4141 caagtaaggt acaaagtggt gaattccggc aattcgatat caagcttatc gataatcaac
     4201 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     4261 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     4321 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     4381 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     4441 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     4501 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     4561 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg
     4621 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     4681 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     4741 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc
     4801 gagtaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca
     4861 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga
     4921 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     4981 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     5041 tttgccgcca gaacacagga ccggttctag agcgctgcca ccatgtctag gctggacaag
     5101 agcaaagtca taaacggagc tctggaatta ctcaatggtg tcggtatcga aggcctgacg
     5161 acaaggaaac tcgctcaaaa gctgggagtt gagcagccta ccctgtactg gcacgtgaag
     5221 aacaagcggg ccctgctcga tgccctgcca atcgagatgc tggacaggca tcatacccac
     5281 ttctgccccc tggaaggcga gtcatggcaa gactttctgc ggaacaacgc caagtcatac
     5341 cgctgtgctc tcctctcaca tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca
     5401 gagaaacagt acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc
     5461 ctggagaacg cactgtacgc tctgtccgcc gtgggccact ttacactggg ctgcgtattg
     5521 gaggaacagg agcatcaagt agcaaaagag gaaagagaga cacctaccac cgattctatg
     5581 cccccacttc tgagacaagc aattgagctg ttcgaccggc agggagccga acctgccttc
     5641 cttttcggcc tggaactaat catatgtggc ctggagaaac agctaaagtg cgaaagcggc
     5701 gggccgaccg acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac
     5761 gattttgacc ttgacatgct ccccggggga tccggcgcaa caaacttctc tctgctgaaa
     5821 caagccggag atgtcgaaga gaatcctgga ccgatggcca agcctttgtc tcaagaagaa
     5881 tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac
     5941 agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat
     6001 cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca
     6061 gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc
     6121 tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgaag
     6181 gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg
     6241 tgggagggct aaggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     6301 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat
     6361 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc
     6421 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
     6481 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag
     6541 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct
     6601 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga
     6661 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     6721 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     6781 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc
     6841 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     6901 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     6961 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     7021 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     7081 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     7141 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     7201 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     7261 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     7321 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     7381 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     7441 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     7501 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     7561 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     7621 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     7681 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     7741 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     7801 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     7861 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     7921 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
     7981 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
     8041 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
     8101 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
     8161 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
     8221 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
     8281 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
     8341 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat
     8401 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     8461 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     8521 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     8581 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     8641 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     8701 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     8761 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     8821 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     8881 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     8941 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     9001 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     9061 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     9121 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     9181 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     9241 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     9301 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     9361 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     9421 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     9481 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     9541 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     9601 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     9661 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     9721 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     9781 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     9841 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     9901 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     9961 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
    10021 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
    10081 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
    10141 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
    10201 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
    10261 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
    10321 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
    10381 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
    10441 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
    10501 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
    10561 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
    10621 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
    10681 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
    10741 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
    10801 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
    10861 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
    10921 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
    10981 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
    11041 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
    11101 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
    11161 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
    11221 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
    11281 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
    11341 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
    11401 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
    11461 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
    11521 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
    11581 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
    11641 aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga
    11701 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
    11761 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
    11821 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
    11881 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
    11941 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
    12001 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
    12061 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
    12121 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
    12181 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
    12241 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
    12301 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
    12361 aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
    12421 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
    12481 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
    12541 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
    12601 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
    12661 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
    12721 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
    12781 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt
    12841 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
    12901 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
    12961 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
    13021 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
    13081 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
    13141 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga
    13201 gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat
    13261 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
    13321 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
    13381 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggttaattaa
//
