LOCUS       sequence-453995-       14341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RfxCas13d (NLS-RfxCas13d-NLS) with 2A-EGFP and 2A-mCD4 for RNA 
            knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14341)
  AUTHORS   El Kassem G, Hillmer J, Boettcher M
  TITLE     Evaluation of Cas13d as a tool for genetic interaction mapping.
  JOURNAL   Nat Commun. 2025 Feb 14;16(1):1631. doi: 10.1038/s41467-025-56747-4.
  PUBMED    39952934
REFERENCE   2  (bases 1 to 14341)
  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 
            Commun."; date: "2025-02-14"; volume: "16(1):1631. doi"; pages: " 
            10.1038/s41467-025-56747-4"
COMMENT     Sequence Label: pXR001-mCD4: EF1a-RfxCas13d-P2A-mCD4-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..14341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    61..178
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        303..1481
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          534..1472
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1429..1449
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1500..1520
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1530..4427
                     /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             4437..4457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4467..4493
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4503..4559
                     /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             5859..5912
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(5913..5915,5916..6629)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5913..5915,5916..6626)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5955..5976)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6216..6235)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6563..6584
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    6654..7242
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6707..6727)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7125..7136)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7245..7261)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7246..7262)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             7767..7947
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(7973..7990)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7979..8203
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8249..8677
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8336..8355)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8546..8567
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8686..8706)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8691..9020
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8871..9006
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8933..8952
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9068..9115
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9134..9508
                     /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    9638..9759
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9675..9694)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9729..9748
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9808..9824)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9808..9824)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9821..9843)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9832..9848
                     /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(9856..9862,9863..9880,9881..9886))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9901..9922
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10039..10056)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10210..10798)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10290..10309)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10969..11760,11761..11829))
                     /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     11592..11611
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11830..11934)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12009..12028)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12200..12579
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        12581..12779
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     12734..12754
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             12797..12977
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    13024..13149
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    13642..13875
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             14060..14104
                     /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"
ORIGIN
        1 gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa
       61 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
      121 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
      181 ggtttattac agggacagca gagatccagt ttggttaatt aatgcaaaga tggataaagt
      241 tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa
      301 ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
      361 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
      421 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
      481 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
      541 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
      601 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
      661 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
      721 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
      781 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
      841 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
      901 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
      961 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
     1021 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     1081 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     1141 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     1201 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     1261 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     1321 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     1381 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     1441 cagtggttca aagttttttt cttccatttc aggtgtcgtg acgtacggcc accatgagcc
     1501 ccaagaagaa gagaaaggtg gaggccagca tcgaaaaaaa aaagtccttc gccaagggca
     1561 tgggcgtgaa gtccacactc gtgtccggct ccaaagtgta catgacaacc ttcgccgaag
     1621 gcagcgacgc caggctggaa aagatcgtgg agggcgacag catcaggagc gtgaatgagg
     1681 gcgaggcctt cagcgctgaa atggccgata aaaacgccgg ctataagatc ggcaacgcca
     1741 aattcagcca tcctaagggc tacgccgtgg tggctaacaa ccctctgtat acaggacccg
     1801 tccagcagga tatgctcggc ctgaaggaaa ctctggaaaa gaggtacttc ggcgagagcg
     1861 ctgatggcaa tgacaatatt tgtatccagg tgatccataa catcctggac attgaaaaaa
     1921 tcctcgccga atacattacc aacgccgcct acgccgtcaa caatatctcc ggcctggata
     1981 aggacattat tggattcggc aagttctcca cagtgtatac ctacgacgaa ttcaaagacc
     2041 ccgagcacca tagggccgct ttcaacaata acgataagct catcaacgcc atcaaggccc
     2101 agtatgacga gttcgacaac ttcctcgata accccagact cggctatttc ggccaggcct
     2161 ttttcagcaa ggagggcaga aattacatca tcaattacgg caacgaatgc tatgacattc
     2221 tggccctcct gagcggactg aggcactggg tggtccataa caacgaagaa gagtccagga
     2281 tctccaggac ctggctctac aacctcgata agaacctcga caacgaatac atctccaccc
     2341 tcaactacct ctacgacagg atcaccaatg agctgaccaa ctccttctcc aagaactccg
     2401 ccgccaacgt gaactatatt gccgaaactc tgggaatcaa ccctgccgaa ttcgccgaac
     2461 aatatttcag attcagcatt atgaaagagc agaaaaacct cggattcaat atcaccaagc
     2521 tcagggaagt gatgctggac aggaaggata tgtccgagat caggaaaaat cataaggtgt
     2581 tcgactccat caggaccaag gtctacacca tgatggactt tgtgatttat aggtattaca
     2641 tcgaagagga tgccaaggtg gctgccgcca ataagtccct ccccgataat gagaagtccc
     2701 tgagcgagaa ggatatcttt gtgattaacc tgaggggctc cttcaacgac gaccagaagg
     2761 atgccctcta ctacgatgaa gctaatagaa tttggagaaa gctcgaaaat atcatgcaca
     2821 acatcaagga atttagggga aacaagacaa gagagtataa gaagaaggac gcccctagac
     2881 tgcccagaat cctgcccgct ggccgtgatg tttccgcctt cagcaaactc atgtatgccc
     2941 tgaccatgtt cctggatggc aaggagatca acgacctcct gaccaccctg attaataaat
     3001 tcgataacat ccagagcttc ctgaaggtga tgcctctcat cggagtcaac gctaagttcg
     3061 tggaggaata cgcctttttc aaagactccg ccaagatcgc cgatgagctg aggctgatca
     3121 agtccttcgc tagaatggga gaacctattg ccgatgccag gagggccatg tatatcgacg
     3181 ccatccgtat tttaggaacc aacctgtcct atgatgagct caaggccctc gccgacacct
     3241 tttccctgga cgagaacgga aacaagctca agaaaggcaa gcacggcatg agaaatttca
     3301 ttattaataa cgtgatcagc aataaaaggt tccactacct gatcagatac ggtgatcctg
     3361 cccacctcca tgagatcgcc aaaaacgagg ccgtggtgaa gttcgtgctc ggcaggatcg
     3421 ctgacatcca gaaaaaacag ggccagaacg gcaagaacca gatcgacagg tactacgaaa
     3481 cttgtatcgg aaaggataag ggcaagagcg tgagcgaaaa ggtggacgct ctcacaaaga
     3541 tcatcaccgg aatgaactac gaccaattcg acaagaaaag gagcgtcatt gaggacaccg
     3601 gcagggaaaa cgccgagagg gagaagttta aaaagatcat cagcctgtac ctcaccgtga
     3661 tctaccacat cctcaagaat attgtcaata tcaacgccag gtacgtcatc ggattccatt
     3721 gcgtcgagcg tgatgctcaa ctgtacaagg agaaaggcta cgacatcaat ctcaagaaac
     3781 tggaagagaa gggattcagc tccgtcacca agctctgcgc tggcattgat gaaactgccc
     3841 ccgataagag aaaggacgtg gaaaaggaga tggctgaaag agccaaggag agcattgaca
     3901 gcctcgagag cgccaacccc aagctgtatg ccaattacat caaatacagc gacgagaaga
     3961 aagccgagga gttcaccagg cagattaaca gggagaaggc caaaaccgcc ctgaacgcct
     4021 acctgaggaa caccaagtgg aatgtgatca tcagggagga cctcctgaga attgacaaca
     4081 agacatgtac cctgttcaga aacaaggccg tccacctgga agtggccagg tatgtccacg
     4141 cctatatcaa cgacattgcc gaggtcaatt cctacttcca actgtaccat tacatcatgc
     4201 agagaattat catgaatgag aggtacgaga aaagcagcgg aaaggtgtcc gagtacttcg
     4261 acgctgtgaa tgacgagaag aagtacaacg ataggctcct gaaactgctg tgtgtgcctt
     4321 tcggctactg tatccccagg tttaagaacc tgagcatcga ggccctgttc gataggaacg
     4381 aggccgccaa gttcgacaag gagaaaaaga aggtgtccgg caattccgga tccggaccta
     4441 agaaaaagag gaaggtggcg gccgcttacc catacgatgt tccagattac gctggatccg
     4501 gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt cgaagagaat cctggaccgc
     4561 cggacatgtg ccgagccatc tctcttaggc gcttgctgct gctgctgctg cagctgtcac
     4621 aactcctagc tgtcactcaa gggaagacgc tggtgctggg gaaggaaggg gaatcagcag
     4681 aactgccctg cgagagttcc cagaagaaga tcacagtctt cacctggaag ttctctgacc
     4741 agaggaagat tctggggcag catggcaaag gtgtattaat tagaggaggt tcgccttcgc
     4801 agtttgatcg ttttgattcc aaaaaagggg catgggagaa aggatcgttt cctctcatca
     4861 tcaataaact taagatggaa gactctcaga cttatatctg tgagctggag aacaggaaag
     4921 aggaggtgga gttgtgggtg ttcaaagtga ccttcagtcc gggtaccagc ctgttgcaag
     4981 ggcagagcct gaccctgacc ttggatagca actctaaggt ctctaacccc ttgacagagt
     5041 gcaaacacaa aaagggtaaa gttgtcagtg gttccaaagt tctctccatg tccaacctaa
     5101 gggttcagga cagcgacttc tggaactgca ccgtgaccct ggaccagaaa aagaactggt
     5161 tcggcatgac actctcagtg ctgggttttc agagcacagc tatcacggcc tataagagtg
     5221 agggagagtc agcggagttc tccttcccac tcaactttgc agaggaaaac gggtggggag
     5281 agctgatgtg gaaggcagag aaggattctt tcttccagcc ctggatctcc ttctccataa
     5341 agaacaaaga ggtgtccgta caaaagtcca ccaaagacct caagctccag ctgaaggaaa
     5401 cgctcccact caccctcaag ataccccagg tctcgcttca gtttgctggt tctggcaacc
     5461 tgactctgac tctggacaaa gggacactgc atcaggaagt gaacctggtg gtgatgaaag
     5521 tggctcagct caacaatact ttgacctgtg aggtgatggg acctacctct cccaagatga
     5581 gactgaccct gaagcaggag aaccaggagg ccagggtctc tgaggagcag aaagtagttc
     5641 aagtggtggc ccctgagaca gggctgtggc agtgtctact gagtgaaggt gataaggtca
     5701 agatggactc caggatccag gttttatcca gaggggtgaa ccagacagtg ttcctggctt
     5761 gcgtgctggg tggctccttc ggctttctgg gtttccttgg gctctgcatc ctctgctgtg
     5821 tcaggtgccg gcaccaacag cgccagggcg gcagtggaga gggcagagga agtctgctaa
     5881 catgcggtga cgtcgaggag aatcctggcc cagtgagcaa gggcgaggag ctgttcaccg
     5941 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     6001 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     6061 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     6121 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     6181 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     6241 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     6301 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     6361 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     6421 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     6481 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     6541 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     6601 ctctcggcat ggacgagctg tacaagtaag aattcgatat caagcttatc gataatcaac
     6661 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     6721 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     6781 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     6841 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     6901 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     6961 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     7021 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg
     7081 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     7141 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     7201 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc
     7261 gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt
     7321 gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac
     7381 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     7441 ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggatct
     7501 accacacaca aggctacttc cctgattggc agaactacac accagggcca gggatcagat
     7561 atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag aaggtagaag
     7621 aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat gggatggatg
     7681 acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt catcacatgg
     7741 cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc tgagcctggg
     7801 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     7861 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     7921 tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc agcctcgact
     7981 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     8041 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     8101 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     8161 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     8221 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     8281 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     8341 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     8401 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     8461 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     8521 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     8581 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     8641 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     8701 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     8761 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     8821 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     8881 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     8941 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     9001 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     9061 agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     9121 gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt
     9181 cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga
     9241 cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca
     9301 ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc
     9361 cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga
     9421 gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt
     9481 gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt ccaccgccgc
     9541 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     9601 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     9661 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     9721 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     9781 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     9841 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     9901 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     9961 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
    10021 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
    10081 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
    10141 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
    10201 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
    10261 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
    10321 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
    10381 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
    10441 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
    10501 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
    10561 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
    10621 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
    10681 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
    10741 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
    10801 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
    10861 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
    10921 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
    10981 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
    11041 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
    11101 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
    11161 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
    11221 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
    11281 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
    11341 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
    11401 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
    11461 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
    11521 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
    11581 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
    11641 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
    11701 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
    11761 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
    11821 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
    11881 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
    11941 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
    12001 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
    12061 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
    12121 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
    12181 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
    12241 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
    12301 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
    12361 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
    12421 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
    12481 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
    12541 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
    12601 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
    12661 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
    12721 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcagc
    12781 gcgttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
    12841 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
    12901 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
    12961 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
    13021 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
    13081 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
    13141 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
    13201 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
    13261 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
    13321 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
    13381 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
    13441 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt
    13501 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
    13561 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
    13621 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
    13681 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
    13741 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
    13801 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
    13861 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
    13921 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
    13981 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
    14041 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
    14101 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
    14161 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
    14221 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga
    14281 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt
    14341 a
//
