LOCUS       sequence-456688-       13440 bp    DNA     circular SYN 15-DEC-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)
  AUTHORS   Hart SK, Muller S, Wessels HH, Mendez-Mancilla A, Drabavicius G, 
            Choi O, Sanjana NE
  TITLE     Precise RNA targeting with CRISPR-Cas13d.
  JOURNAL   Nat Biotechnol. 2025 Feb 11. doi: 10.1038/s41587-025-02558-3.
  PUBMED    39934271
REFERENCE   2  (bases 1 to 13440)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 Feb 11. doi: 10.1038/s41587-025-02558-3."
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"
     CDS             662..718
                     /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             719..1117
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             1509..1689
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(1715..1732)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1721..1945
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1991..2419
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2078..2097)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2288..2309
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2428..2448)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2433..2762
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2613..2748
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2675..2694
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2810..2857
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2876..3250
                     /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    3380..3501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3417..3436)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3471..3490
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3550..3566)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3550..3566)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3563..3585)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3574..3590
                     /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(3598..3604,3605..3622,3623..3628))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3643..3664
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3781..3798)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3952..4540)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4032..4051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4711..5502,5503..5571))
                     /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     5334..5353
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5572..5676)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5751..5770)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5942..6321
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6323..6521
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6539..6719
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6766..6891
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7384..7617
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7802..7846
                     /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    8144..8261
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    8306..8326
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    8371..8389
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8406..8424
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8442..8460
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8478..8496
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8513..8531
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8549..8567
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        8579..8617
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8614..8638
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             8682..8702
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8712..11609
                     /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             11619..11639
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11649..11675
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             11685..11741
                     /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             11742..12452
                     /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(11887..11905)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     12155..12174
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    12499..13087
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12552..12572)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12970..12981)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(13090..13106)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13091..13107)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        13152..13363
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             join(13388..13440,1..652)
                     /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"
ORIGIN
        1 tggtgtcggt atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca
       61 gcctaccctg tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga
      121 gatgctggac aggcatcata cccacttctg ccccctggaa ggcgagtcat ggcaagactt
      181 tctgcggaac aacgccaagt cataccgctg tgctctcctc tcacatcgcg acggggctaa
      241 agtgcatctc ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc
      301 gttcctgtgt cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg
      361 ccactttaca ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag
      421 agagacacct accaccgatt ctatgccccc acttctgaga caagcaattg agctgttcga
      481 ccggcaggga gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga
      541 gaaacagcta aagtgcgaaa gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga
      601 catgctccca gccgatgccc ttgacgattt tgaccttgac atgctccccg ggggatccgg
      661 cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat
      721 ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa
      781 cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg
      841 catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt
      901 gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa
      961 tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca
     1021 tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg
     1081 tgaattgctg ccctctggtt atgtgtggga gggctaaggt acctttaaga ccaatgactt
     1141 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     1201 ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact
     1261 tccctgattg gcagaactac acaccagggc cagggatcag atatccactg acctttggat
     1321 ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga
     1381 acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat
     1441 tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg
     1501 actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     1561 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     1621 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     1681 tctctagcag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     1741 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1801 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1861 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     1921 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     1981 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2041 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2101 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2161 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2221 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2281 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2341 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2401 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2461 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2521 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2581 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2641 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     2701 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     2761 aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta
     2821 atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc
     2881 caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt
     2941 ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt
     3001 ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac
     3061 cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt
     3121 gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg
     3181 ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga
     3241 gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     3301 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3361 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3421 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3481 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     3541 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3601 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3661 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3721 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3781 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3841 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3901 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3961 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4021 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4081 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4141 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4201 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4261 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4321 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4381 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4441 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4501 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4561 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4621 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4681 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4741 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4801 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4861 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4921 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4981 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5041 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5101 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5161 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5221 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5281 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5341 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5401 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5461 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5521 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5581 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5641 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5701 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     5761 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     5821 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     5881 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     5941 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     6001 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     6061 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     6121 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     6181 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     6241 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     6301 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     6361 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     6421 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     6481 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg
     6541 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     6601 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     6661 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     6721 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
     6781 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
     6841 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
     6901 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     6961 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     7021 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     7081 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     7141 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     7201 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     7261 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     7321 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     7381 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     7441 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     7501 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     7561 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     7621 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     7681 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     7741 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     7801 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     7861 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     7921 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     7981 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     8041 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     8101 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     8161 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     8221 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     8281 gcagagatcc agtttggtta attaacaact ttgtatagaa aagttggctc cgaattcgcc
     8341 cttcaggtcc gaggttctag acgagtttac tccctatcag tgatagagaa cgatgtcgag
     8401 tttactccct atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga
     8461 acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt tatccctatc
     8521 agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagg
     8581 taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc
     8641 aaagggcgaa ttcgacccaa gtttgtacag ccaccatgag ccccaagaag aagagaaagg
     8701 tggaggccag catcgaaaaa aaaaagtcct tcgccaaggg catgggcgtg aagtccacac
     8761 tcgtgtccgg ctccaaagtg tacatgacaa ccttcgccga aggcagcgac gccaggctgg
     8821 aaaagatcgt ggagggcgac agcatcagga gcgtgaatga gggcgaggcc ttcagcgctg
     8881 aaatggccga taaaaacgcc ggctataaga tcggcaacgc caaattcagc catcctaagg
     8941 gctacgccgt ggtggctaac aaccctctgt atacaggacc cgtccagcag gatatgctcg
     9001 gcctgaagga aactctggaa aagaggtact tcggcgagag cgctgatggc aatgacaata
     9061 tttgtatcca ggtgatccat aacatcctgg acattgaaaa aatcctcgcc gaatacatta
     9121 ccaacgccgc ctacgccgtc aacaatatct ccggcctgga taaggacatt attggattcg
     9181 gcaagttctc cacagtgtat acctacgacg aattcaaaga ccccgagcac catagggccg
     9241 ctttcaacaa taacgataag ctcatcaacg ccatcaaggc ccagtatgac gagttcgaca
     9301 acttcctcga taaccccaga ctcggctatt tcggccaggc ctttttcagc aaggagggca
     9361 gaaattacat catcaattac ggcaacgaat gctatgacat tctggccctc ctgagcggac
     9421 tgaggcactg ggtggtccat aacaacgaag aagagtccag gatctccagg acctggctct
     9481 acaacctcga taagaacctc gacaacgaat acatctccac cctcaactac ctctacgaca
     9541 ggatcaccaa tgagctgacc aactccttct ccaagaactc cgccgccaac gtgaactata
     9601 ttgccgaaac tctgggaatc aaccctgccg aattcgccga acaatatttc agattcagca
     9661 ttatgaaaga gcagaaaaac ctcggattca atatcaccaa gctcagggaa gtgatgctgg
     9721 acaggaagga tatgtccgag atcaggaaaa atcataaggt gttcgactcc atcaggacca
     9781 aggtctacac catgatggac tttgtgattt ataggtatta catcgaagag gatgccaagg
     9841 tggctgccgc caataagtcc ctccccgata atgagaagtc cctgagcgag aaggatatct
     9901 ttgtgattaa cctgaggggc tccttcaacg acgaccagaa ggatgccctc tactacgatg
     9961 aagctaatag aatttggaga aagctcgaaa atatcatgca caacatcaag gaatttaggg
    10021 gaaacaagac aagagagtat aagaagaagg acgcccctag actgcccaga atcctgcccg
    10081 ctggccgtga tgtttccgcc ttcagcaaac tcatgtatgc cctgaccatg ttcctggatg
    10141 gcaaggagat caacgacctc ctgaccaccc tgattaataa attcgataac atccagagct
    10201 tcctgaaggt gatgcctctc atcggagtca acgctaagtt cgtggaggaa tacgcctttt
    10261 tcaaagactc cgccaagatc gccgatgagc tgaggctgat caagtccttc gctagaatgg
    10321 gagaacctat tgccgatgcc aggagggcca tgtatatcga cgccatccgt attttaggaa
    10381 ccaacctgtc ctatgatgag ctcaaggccc tcgccgacac cttttccctg gacgagaacg
    10441 gaaacaagct caagaaaggc aagcacggca tgagaaattt cattattaat aacgtgatca
    10501 gcaataaaag gttccactac ctgatcagat acggtgatcc tgcccacctc catgagatcg
    10561 ccaaaaacga ggccgtggtg aagttcgtgc tcggcaggat cgctgacatc cagaaaaaac
    10621 agggccagaa cggcaagaac cagatcgaca ggtactacga aacttgtatc ggaaaggata
    10681 agggcaagag cgtgagcgaa aaggtggacg ctctcacaaa gatcatcacc ggaatgaact
    10741 acgaccaatt cgacaagaaa aggagcgtca ttgaggacac cggcagggaa aacgccgaga
    10801 gggagaagtt taaaaagatc atcagcctgt acctcaccgt gatctaccac atcctcaaga
    10861 atattgtcaa tatcaacgcc aggtacgtca tcggattcca ttgcgtcgag cgtgatgctc
    10921 aactgtacaa ggagaaaggc tacgacatca atctcaagaa actggaagag aagggattca
    10981 gctccgtcac caagctctgc gctggcattg atgaaactgc ccccgataag agaaaggacg
    11041 tggaaaagga gatggctgaa agagccaagg agagcattga cagcctcgag agcgccaacc
    11101 ccaagctgta tgccaattac atcaaataca gcgacgagaa gaaagccgag gagttcacca
    11161 ggcagattaa cagggagaag gccaaaaccg ccctgaacgc ctacctgagg aacaccaagt
    11221 ggaatgtgat catcagggag gacctcctga gaattgacaa caagacatgt accctgttca
    11281 gaaacaaggc cgtccacctg gaagtggcca ggtatgtcca cgcctatatc aacgacattg
    11341 ccgaggtcaa ttcctacttc caactgtacc attacatcat gcagagaatt atcatgaatg
    11401 agaggtacga gaaaagcagc ggaaaggtgt ccgagtactt cgacgctgtg aatgacgaga
    11461 agaagtacaa cgataggctc ctgaaactgc tgtgtgtgcc tttcggctac tgtatcccca
    11521 ggtttaagaa cctgagcatc gaggccctgt tcgataggaa cgaggccgcc aagttcgaca
    11581 aggagaaaaa gaaggtgtcc ggcaattccg gatccggacc taagaaaaag aggaaggtgg
    11641 cggccgctta cccatacgat gttccagatt acgctggaag cggagctact aacttcagcc
    11701 tgctgaagca ggctggagac gtggaggaga accctggacc tatggtgagc aagggcgagg
    11761 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
    11821 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
    11881 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
    11941 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
    12001 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
    12061 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
    12121 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
    12181 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
    12241 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
    12301 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
    12361 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
    12421 ccaccggcgg catggacgag ctgtacaagt aaggtacaaa gtggtgaatt ccggcaattc
    12481 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
    12541 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
    12601 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
    12661 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
    12721 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
    12781 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
    12841 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
    12901 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
    12961 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
    13021 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
    13081 tccccgcatc gataccgtcg acctcgagta ggtcttgaaa ggagtgggaa ttggctccgg
    13141 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
    13201 cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
    13261 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
    13321 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc
    13381 tgccaccatg tctaggctgg acaagagcaa agtcataaac ggagctctgg aattactcaa
//
