LOCUS       sequence-271997-       13101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing high-specificity eSpCas9(1.1) and a 
            gRNA targeting human MIB1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13101)
  AUTHORS   Bauer M, Flatt JW, Seiler D, Cardel B, Emmenlauer M, Boucke K, 
            Suomalainen M, Hemmi S, Greber UF
  TITLE     The E3 Ubiquitin Ligase Mind Bomb 1 Controls Adenovirus Genome 
            Release at the Nuclear Pore Complex.
  JOURNAL   Cell Rep. 2019 Dec 17;29(12):3785-3795.e8. doi: 
            10.1016/j.celrep.2019.11.064.
  PUBMED    31851912
REFERENCE   2  (bases 1 to 13101)
  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"; doi: 
            "10.1016/j.celrep.2019.11"; journalName: "Cell Rep"; date: 
            "2019-12-17- 17"; volume: "29"; issue: "12"; pages: "3785-3795.e8"
COMMENT     Sequence Label: Lenti-eCas9-sgMIB1
FEATURES             Location/Qualifiers
     source          1..13101
                     /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"
     misc_RNA        2877..2952
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3014..3225
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3240..3249
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3249..3314
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3321..3341
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3366..5903,5904..5906,5907..6368,6369..6371,6372..6539,
                     6540..6542,6543..7466)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7467..7514
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7524..7580
                     /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             7581..8177
                     /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     8074..8094
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8193..8781
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8246..8266)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8664..8675)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8853..9086
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9112..9129)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9118..9342
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9388..9816
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9475..9494)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9685..9706
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9825..9845)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9830..10159
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10010..10145
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10072..10091
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10207..10254
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10273..10647
                     /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    10777..10898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10814..10833)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10868..10887
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10947..10963)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10947..10963)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10960..10982)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10971..10987
                     /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(10995..11001,11002..11019,11020..11025))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11040..11061
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11178..11195)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11349..11937)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11429..11448)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12108..12899,12900..12968))
                     /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     12731..12750
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12969..13073)
                     /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 aacaccgttg gcgctcgggt agtgcggttt
     2881 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     2941 ccgagtcggt gcttttttga attcgctagc taggtcttga aaggagtggg aattggctcc
     3001 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     3061 gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     3121 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     3181 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggaccg gttctagatg
     3241 ccaccatgga ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag
     3301 acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag
     3361 cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg
     3421 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc
     3481 ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg
     3541 aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct
     3601 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca
     3661 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg
     3721 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa
     3781 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc
     3841 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg
     3901 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc
     3961 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca
     4021 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa
     4081 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg
     4141 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg
     4201 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca
     4261 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct
     4321 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc
     4381 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg
     4441 ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc
     4501 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc
     4561 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc
     4621 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga
     4681 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca
     4741 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg
     4801 aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata
     4861 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg
     4921 tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc
     4981 tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga
     5041 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa
     5101 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa
     5161 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg
     5221 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg
     5281 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg
     5341 gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc
     5401 tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg
     5461 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc
     5521 tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga
     5581 cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg
     5641 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga
     5701 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc
     5761 ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc
     5821 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc
     5881 atatcgtgcc tcagagcttt ctggccgacg actccatcga caacaaggtg ctgaccagaa
     5941 gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga
     6001 agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc
     6061 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac
     6121 agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga
     6181 acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt
     6241 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca
     6301 actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa
     6361 agtaccctgc gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga
     6421 agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca
     6481 gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagg
     6541 cgcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt
     6601 ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg
     6661 tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga
     6721 tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg
     6781 cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg
     6841 tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg
     6901 actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta
     6961 agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac
     7021 tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca
     7081 gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg
     7141 aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag
     7201 tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata
     7261 agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag
     7321 cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca
     7381 aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga
     7441 tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg
     7501 caaaaaagaa aaagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg
     7561 tcgaagagaa tcctggaccg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     7621 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     7681 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     7741 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     7801 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     7861 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     7921 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc
     7981 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     8041 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     8101 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     8161 gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg
     8221 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     8281 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     8341 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     8401 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     8461 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     8521 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     8581 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     8641 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     8701 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     8761 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga
     8821 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     8881 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     8941 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     9001 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     9061 ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg
     9121 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     9181 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     9241 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     9301 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     9361 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     9421 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     9481 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     9541 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     9601 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     9661 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     9721 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     9781 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     9841 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     9901 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     9961 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
    10021 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
    10081 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
    10141 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
    10201 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
    10261 aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc
    10321 gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac
    10381 gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag
    10441 gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc
    10501 gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag
    10561 atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg
    10621 cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc
    10681 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
    10741 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
    10801 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
    10861 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
    10921 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
    10981 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
    11041 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
    11101 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
    11161 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
    11221 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
    11281 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
    11341 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
    11401 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
    11461 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
    11521 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
    11581 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
    11641 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
    11701 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
    11761 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
    11821 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
    11881 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
    11941 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
    12001 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
    12061 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
    12121 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
    12181 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
    12241 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
    12301 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
    12361 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
    12421 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
    12481 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
    12541 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
    12601 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
    12661 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
    12721 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
    12781 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
    12841 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
    12901 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
    12961 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
    13021 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
    13081 ccccgaaaag tgccacctga c
//
