LOCUS       Exported               12256 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  lentiviral expression of SaCas9 and two pol III promoters for an Sa 
            gRNA and an Sp gRNA. Intended to be used in Stable SpCas9 expressing
            cell lines for dual Cas9 ""Big Papi"" screens. Alt name pXPR207..
ACCESSION   .
VERSION     .
KEYWORDS    pPapi
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12256)
  AUTHORS   Najm FJ, Strand C, Donovan KF, Hegde M, Sanson KR, Vaimberg EW, 
            Sullender ME, Hartenian E, Kalani Z, Fusi N, Listgarten J, Younger 
            ST, Bernstein BE, Root DE, Doench JG
  TITLE     Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.
  JOURNAL   Nat Biotechnol. 2018 Feb;36(2):179-189. doi: 10.1038/nbt.4048. Epub 
            2017 Dec 18.
  PUBMED    29251726
REFERENCE   2  (bases 1 to 12256)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..12256
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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..2848
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     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"
     promoter        complement(2898..3112)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(2958..2977)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        3168..3379
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3387..3396
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3399..3419
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3444..6599
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             6600..6647
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6657..6713
                     /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"
     misc_feature    7342..7930
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7395..7415)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7813..7824)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8008..8241
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8267..8284)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8273..8497
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8543..8971
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8630..8649)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8840..8861
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8980..9000)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8985..9314
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9165..9300
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9227..9246
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9362..9409
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9428..9802
                     /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    9932..10053
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9969..9988)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10023..10042
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10102..10118)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10102..10118)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10115..10137)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10126..10142
                     /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(10150..10180)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10195..10216
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10333..10350)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10504..11092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10584..10603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(11263..12123)
                     /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     11886..11905
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12124..12228)
                     /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 attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acgcgttttt tgcgcgcaaa
     2881 aacgtctccg ggaaagagtg gtctcataca gaacttataa gattcccaaa tccaaagaca
     2941 tttcacgttt atggtgattt cccagaacac atagcgacat gcaaatattg cagggcgcca
     3001 ctcccctgtc cctcacagcc atcttcctgc cagggcgcac gcgcgctggg tgttcccgcc
     3061 tagtgacact gggcccgcga ttccttggag cgggttgatg acgtcagcgt tcgaattcgc
     3121 tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac
     3181 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag
     3241 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     3301 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     3361 gtttgccgcc agaacacagg accggtgcca ccatggcacc caagaagaag cgcaaagttg
     3421 gcatccacgg cgtgcctgct gccaagcgca actacatcct gggcctggac attggcatca
     3481 cctcagttgg ctatggcatc atcgactacg agacccggga cgtcatcgac gctggcgtcc
     3541 gcctcttcaa ggaggccaac gtcgagaaca acgagggccg gcggtccaag cgcggggccc
     3601 gccggctcaa gcgccgccgc cgccaccgca tccagcgcgt gaaaaagttg ctcttcgact
     3661 acaacttgct caccgaccac tccgagctgt ctggcatcaa cccgtacgag gcccgggtca
     3721 agggcctctc gcaaaagttg tccgaggagg agttctctgc tgccctgctg cacctggcca
     3781 agcgccgcgg ggtccacaac gtcaacgaag ttgaggagga caccggcaac gaactttcca
     3841 ccaaggagca gatctcccgc aactccaagg ccctggagga gaagtacgtc gcggagctcc
     3901 aacttgagcg cctcaagaag gacggcgagg tgcgcggctc catcaaccgc ttcaagacct
     3961 ccgactacgt caaggaggcc aagcagctgc tcaaggtcca gaaggcctac caccaacttg
     4021 accagtcctt catcgacacc tacatcgacc tgctggagac ccgccgcacc tactacgagg
     4081 ggcctggcga gggctctccc ttcggctgga aggacatcaa ggagtggtac gagatgctca
     4141 tgggccactg cacctacttc ccggaggagc tgcgctcggt caagtacgcc tacaacgccg
     4201 acctctacaa cgcgctcaac gacctcaaca acttggtcat cacccgcgac gagaacgaaa
     4261 agttggagta ctacgaaaag ttccagatca tcgaaaacgt cttcaagcag aagaagaagc
     4321 caactttgaa gcagatcgcc aaggagatcc tggtcaacga ggaggacatc aagggctacc
     4381 gggtcacctc caccggcaag cccgagttca ccaacttaaa ggtctaccat gacatcaagg
     4441 acatcaccgc ccgcaaggag atcatcgaga acgcggagct gctggaccag atcgccaaga
     4501 tcctcaccat ctaccaaagt tccgaggaca tccaggagga gctgaccaac ttaaactcgg
     4561 agcttaccca ggaggagatc gagcagatct ccaacttaaa gggctacact ggcacccaca
     4621 acttgtccct caaggccatc aacttgatcc tggacgaact ttggcatacc aacgacaacc
     4681 agatcgccat cttcaaccga ctaaagttgg tgcccaagaa agttgacctg tcccaacaga
     4741 aggagatccc aactaccctg gtcgacgact tcatcctctc gcccgtggtc aagcgcagct
     4801 tcatccagtc catcaaggtc atcaacgcca tcatcaagaa gtacggcctg cccaacgaca
     4861 tcatcatcga gttggcacgc gagaagaact ccaaggatgc ccagaagatg atcaacgaga
     4921 tgcagaagcg caaccgccag accaacgagc gcatcgagga gatcatccgc accaccggca
     4981 aggagaacgc caagtacctg atcgagaaga taaagttgca tgacatgcag gagggcaagt
     5041 gcctctactc cctggaggcc atccccctcg aggacctgct caacaacccc ttcaactacg
     5101 aagttgacca catcatcccg cgctcggtgt ccttcgacaa cagcttcaac aacaaggtcc
     5161 tggtcaagca ggaggagaac tccaagaagg gcaaccggac ccccttccag tacctgtcct
     5221 cctcggactc caagatctcc tacgaaactt tcaagaagca catcctcaac ttggcaaagg
     5281 gcaagggccg catctccaag accaagaagg agtacctgct ggaggagcgc gacatcaacc
     5341 gcttctcggt gcagaaggac ttcatcaacc gcaacctggt ggacacccgc tacgcaaccc
     5401 gtggcctcat gaacctgctg cgcagctact tccgggtcaa caacttggac gtcaaagtta
     5461 agtcgatcaa cggtggcttc accagcttcc tgcgccgcaa gtggaagttc aagaaggagc
     5521 gcaacaaggg ctacaagcac catgcggagg acgccctcat catcgccaac gccgacttca
     5581 tcttcaagga gtggaaaaag ttggacaagg ccaagaaggt catggagaac cagatgttcg
     5641 aggagaagca ggccgagtcc atgccggaga tcgagacaga gcaggagtac aaggagatct
     5701 tcatcacgcc gcaccagatc aagcacatca aggacttcaa ggactacaag tactcccacc
     5761 gcgtggacaa gaagcccaac cgcgagctca tcaacgacac cctctactcc acccgcaagg
     5821 acgacaaggg caacaccctc atcgtcaaca acttaaacgg cctctacgac aaggacaacg
     5881 acaagttgaa aaagttgatc aacaagagcc ccgaaaagtt gctcatgtac caccatgacc
     5941 cgcagacgta ccaaaagttg aagttgatca tggagcagta cggtgacgag aagaacccgc
     6001 tctacaagta ctacgaggag accggcaact acctcaccaa gtactccaag aaggacaacg
     6061 gcccggtcat caagaagatc aagtactacg gcaacaagtt gaatgcccac ctggacatca
     6121 ccgacgacta ccccaactcc cgcaacaaag ttgtaaagtt gtcgctaaag ccgtaccgct
     6181 tcgacgtcta cctggacaac ggcgtctaca agttcgtgac cgtcaagaac ctggacgtca
     6241 tcaagaagga gaactactac gaggtcaact ccaagtgcta cgaggaggcc aaaaagttga
     6301 agaagatctc caaccaggcc gagttcatcg ccagcttcta caacaacgac ctcatcaaga
     6361 tcaacggcga gctctaccgc gtcatcgggg tcaacaacga cctgctgaac cgcatcgagg
     6421 tcaacatgat cgacatcacc taccgcgagt acctcgagaa catgaacgac aagcgcccgc
     6481 ctcgcatcat caagaccatc gcctccaaga cccagtccat caagaagtac tccaccgaca
     6541 tccttggcaa cttgtacgag gtcaagagca agaagcaccc tcagatcatc aagaagggca
     6601 agcgccctgc tgccaccaag aaggctggcc aggccaagaa gaagaaggga tccggggcca
     6661 ccaacttttc cctgctgaag caggctggcg acgtcgagga gaaccctggc cctggctccg
     6721 gaaccgagta caagccaact gtccgcttgg caacccgcga cgatgtgccc agggccgtcc
     6781 gcacgctggc tgctgccttc gccgactacc cagccacccg ccacaccgtg gacccggacc
     6841 gccacatcga gcgcgtcacc gagctgcagg agctcttcct cacccgagtt ggccttgaca
     6901 ttggcaaggt ctgggtggcg gacgacggtg ctgccgtggc cgtctggacc acccccgagt
     6961 cggtcgaggc tggcgccgtc ttcgccgaga tcgggccgcg catggccgag ctttcgggct
     7021 cccggttggc tgcccaacag cagatggagg ggctgttggc accccaccgc cccaaggagc
     7081 cggcctggtt cctggccaca gttggtgtct ctcccgacca ccagggcaag ggcctgggca
     7141 gcgccgtggt gctgcctggg gtcgaggctg ccgagcgcgc tggcgtccct gccttcctgg
     7201 agacctctgc ccctcgcaac ttgcccttct acgagcgcct gggcttcacc gtcaccgcgg
     7261 acgtcgaggt gcccgagggg ccgcgcacct ggtgcatgac ccgcaagcct ggggcatgaa
     7321 tctagaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact
     7381 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     7441 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     7501 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     7561 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     7621 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     7681 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     7741 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     7801 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     7861 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     7921 gcctccccgc agcgctgtcg actttaagac caatgactta caaggcagct gtagatctta
     7981 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     8041 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     8101 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     8161 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     8221 cagtgtggaa aatctctagc agggcccgtt taaacccgct gatcagcctc gactgtgcct
     8281 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     8341 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     8401 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     8461 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     8521 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     8581 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     8641 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     8701 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     8761 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     8821 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     8881 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     8941 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     9001 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     9061 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     9121 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     9181 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     9241 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     9301 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg
     9361 tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa
     9421 ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg
     9481 agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt
     9541 cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt
     9601 gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg
     9661 gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg
     9721 cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt
     9781 cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta
     9841 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     9901 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     9961 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
    10021 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
    10081 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
    10141 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
    10201 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
    10261 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
    10321 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
    10381 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
    10441 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
    10501 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
    10561 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
    10621 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
    10681 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
    10741 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
    10801 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
    10861 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
    10921 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
    10981 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
    11041 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
    11101 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
    11161 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
    11221 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
    11281 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
    11341 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
    11401 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
    11461 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
    11521 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
    11581 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
    11641 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
    11701 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
    11761 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
    11821 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
    11881 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
    11941 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
    12001 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
    12061 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
    12121 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
    12181 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
    12241 aaaagtgcca cctgac
//
