LOCUS       sequence-266641-       12364 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses PguCas13b in mammalian cells, localized in the nucleus. 
            For cloning of guide RNAs compatible with PguCas13b. Contains a 3' 
            direct repeat. Clone using BsmBI. F overhang cacc. R overhang caac.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12364)
  AUTHORS   Wessels HH, Mendez-Mancilla A, Guo X, Legut M, Daniloski Z, Sanjana 
            NE
  TITLE     Massively parallel Cas13 screens reveal principles for guide RNA 
            design.
  JOURNAL   Nat Biotechnol. 2020 Jun;38(6):722-727. doi: 
            10.1038/s41587-020-0456-9. Epub 2020 Mar 16.
  PUBMED    32518401
REFERENCE   2  (bases 1 to 12364)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41587-020-0456-9"; journalName: "Nat Biotechnol"; date: 
            "2020-06"; volume: "38"; issue: "6"; pages: "722-727"
COMMENT     Sequence Label: pLentiRNACRISPR_001 -
            hU6-DR_BsmBI-EFS-PguCas1...
FEATURES             Location/Qualifiers
     source          1..12364
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(2878..2889)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        2969..3180
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             6730..6777
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6787..6843
                     /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             6844..7440
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7337..7357
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7456..8044
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7509..7529)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7927..7938)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8116..8349
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8375..8392)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8381..8605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8651..9079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8738..8757)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8948..8969
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9088..9108)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9093..9422
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9273..9408
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9335..9354
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9470..9517
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9536..9910
                     /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    10040..10161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10077..10096)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10131..10150
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10210..10226)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10210..10226)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10223..10245)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10234..10250
                     /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(10258..10264,10265..10282,10283..10288))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10303..10324
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10441..10458)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10612..11200)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10692..10711)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11371..12162,12163..12231))
                     /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     11994..12013
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12232..12336)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccgaga cggacgtctc tgttggatct
     2881 accctctatt tgaagggtac acacaacttt tttgaattcg ctagctaggt cttgaaagga
     2941 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     3001 aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac
     3061 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     3121 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     3181 gaccggttct agagcgctgc caccatgaca gagcagagcg agaggcccta caacggcacc
     3241 tactacaccc tggaagataa gcacttctgg gccgcctttc tgaacctggc cagacacaac
     3301 gcctacatca ccctgacaca catcgaccgg cagctggcct acagcaaggc cgacatcacc
     3361 aacgaccagg acgtgctgag cttcaaggcc ctgtggaaga acttcgacaa cgacctggaa
     3421 agaaagagcc ggctgcggag cctgatcctg aagcacttca gctttctgga aggcgccgct
     3481 tacggcaaga agctgttcga gtctaagagc agcggcaaca agagcagcaa gaacaaagag
     3541 ctgaccaaga aagagaaaga ggaactgcag gccaacgctc tgagcctgga caacctgaag
     3601 tctatcctgt tcgacttcct gcagaagctg aaggacttcc ggaactacta cagccactac
     3661 cggcacagcg gaagcagcga gctgcctctg ttcgacggca acatgctgca gcggctgtac
     3721 aacgtgttcg acgtgtccgt gcagcgcgtg aagatcgacc acgagcacaa cgatgaggtg
     3781 gaccctcact accacttcaa ccacctcgtg cggaagggca agaaggacag atacggccac
     3841 aacgacaacc ccagcttcaa gcaccacttc gtggacggcg aaggcatggt tacagaagcc
     3901 ggcctgctgt tcttcgtgtc cctgttcctg gaaaagcggg acgccatctg gatgcagaag
     3961 aagatccggg gcttcaaagg cggcaccgag acataccagc agatgaccaa cgaggtgttc
     4021 tgccggtcca gaatcagcct gcctaagctg aagctggaaa gcctgcggat ggacgactgg
     4081 atgctgctgg acatgctgaa cgaactcgtg cggtgcccca agcctctgta cgacagactg
     4141 agagaggacg accgggcctg ctttagagtg cccgtggata tcctgcctga cgaggacgat
     4201 acagatggcg gcggagagga ccccttcaag aataccctcg tccggcacca ggacagattc
     4261 ccttacttcg ccctgcggta cttcgacctg aagaaggtgt tcaccagcct gcggttccac
     4321 atcgatctgg gcacctacca ctttgccatc tacaagaaga tgatcggcga gcagcccgag
     4381 gacagacacc tgaccagaaa cctgtacggc ttcggccgga tccaggactt cgccgaagaa
     4441 cacagacccg aggaatggaa gcggctcgtc agagatctgg actacttcga gacaggcgac
     4501 aagccctaca tcagccagac aagcccacac taccacatcg agaagggaaa gatcggcctg
     4561 agattcatgc ccgagggcca gcatctttgg ccctctccag aagtgggcac caccagaacc
     4621 ggcagatcta agtacgccca ggacaagaga ctgaccgccg aggcctttct gtctgtgcac
     4681 gagctgatgc ctatgatgtt ctactacttc ctgctgcgcg agaagtacag cgaggaagtg
     4741 tctgccgaga gagtgcaggg cagaatcaag cgcgtgatcg aggatgtgta cgccgtgtac
     4801 gatgcctttg ccagggacga gatcaacacc cgggatgagc tggatgcctg cctggccgat
     4861 aagggcatca gaagaggcca tctgcctcgc cagatgatcg ccatcctgag ccaagagcac
     4921 aaggacatgg aagagaagat tcggaagaaa ctgcaagaga tgatggccga caccgaccac
     4981 cggctggata tgctggatag acagaccgac cggaagatca gaatcggccg gaagaatgcc
     5041 ggactgccta agagcggagt gatcgccgat tggctcgtgc gggacatgat gagatttcag
     5101 cccgtggcca aggacgccag cggaaagcct ctgaacaact ccaaggccaa cagcaccgag
     5161 taccggatgc tgcagagagc cctggctctg tttggaggcg agaaagagag gctgacccca
     5221 tacttccggc agatgaatct gaccggcgga aacaaccctc atccttttct gcacgaaact
     5281 cgctgggaga gccacaccaa catcctgtcc ttttaccggt cctacctgag agcccgcaag
     5341 gccttcctcg aaaggatcgg cagatccgac agagtggaaa accggccatt tctgctgctg
     5401 aaagagccca agaccgacag acagacactg gtggctggat ggaagggcga gttccatctg
     5461 ccaagaggca tcttcacaga ggccgtgcgg gattgcctga tcgagatggg acacgatgaa
     5521 gtggccagct acaaagaagt gggattcatg gccaaggccg tgccactgta ctttgagaga
     5581 gcctgcgagg acagggtgca gcccttttac gacagcccct tcaacgtggg caacagcctg
     5641 aagcctaaga agggcagatt cctgagcaaa gaggaacggg ccgaagagtg ggagcggggc
     5701 aaagagagat tccgggatct cgaagcctgg tcctatagcg ccgccagaag aatcgaggac
     5761 gcctttgccg gcatcgagta tgctagcccc ggaaacaaga agaaaatcga gcagctcctg
     5821 cgggacctga gcctgtggga agcctttgag agcaagctga aagtgcgggc cgaccggatc
     5881 aatctggcca agctgaagaa agaaatcctg gaagcccaag aacaccccta ccacgacttc
     5941 aagagctggc agaagttcga gcgcgagctg cggctggtca agaaccagga tatcatcacc
     6001 tggatgatgt gccgcgacct gatggaagaa aacaaggtgg aaggcctgga caccggcaca
     6061 ctgtacctga aggacatcag acccaacgtg caagagcagg gcagcctgaa cgtgctgaac
     6121 agagtgaagc ccatgagact gcccgtggtg gtgtacagag ccgatagcag aggccacgtg
     6181 cacaaagaag aagcccctct ggccaccgtg tacatcgagg aaagagacac caagctgctg
     6241 aagcagggca acttcaagtc cttcgtgaag gacaggcggc tgaacggcct gttctccttc
     6301 gttgatactg gcggactggc catggaacag taccccatct ccaagctgag agtggaatac
     6361 gagctggcca agtaccagac cgccagagtg tgtgtgttcg agctgaccct gagactggaa
     6421 gagtccctgc tgacccggta tcctcatctg cccgacgaga gcttcagaga gatgctggaa
     6481 tcttggagcg accctctgct ggccaaatgg cctgaactgc acggaaaagt gcggctgctg
     6541 atcgccgtgc ggaatgcctt tagccacaat cagtacccta tgtacgacga ggccgtgttc
     6601 agctccatcc ggaagtacga tcctagcagc cccgacgcca tcgaagagag aatgggcctg
     6661 aatatcgccc accgcctgtc tgaggaagtg aagcaggcca aagaaaccgt ggaacggatc
     6721 attcaggcca agcgacctgc cgccacaaag aaggctggac aggctaagaa gaagaaagga
     6781 tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga gaatcctgga
     6841 ccgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     6901 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     6961 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     7021 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     7081 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     7141 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     7201 cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc
     7261 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     7321 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     7381 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     7441 acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     7501 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     7561 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     7621 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     7681 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     7741 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     7801 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     7861 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     7921 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     7981 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     8041 ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     8101 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     8161 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     8221 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     8281 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     8341 tctctagcag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     8401 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     8461 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     8521 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     8581 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     8641 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     8701 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     8761 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     8821 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     8881 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     8941 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     9001 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     9061 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     9121 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     9181 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     9241 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     9301 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     9361 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     9421 aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta
     9481 atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc
     9541 caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt
     9601 ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt
     9661 ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac
     9721 cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt
     9781 gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg
     9841 ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga
     9901 gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     9961 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
    10021 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
    10081 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
    10141 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
    10201 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
    10261 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
    10321 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
    10381 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
    10441 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
    10501 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
    10561 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
    10621 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
    10681 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
    10741 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
    10801 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
    10861 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
    10921 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
    10981 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
    11041 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
    11101 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
    11161 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
    11221 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
    11281 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
    11341 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
    11401 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
    11461 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
    11521 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
    11581 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
    11641 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
    11701 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
    11761 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
    11821 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
    11881 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
    11941 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
    12001 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
    12061 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
    12121 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
    12181 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
    12241 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
    12301 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
    12361 tgac
//
