LOCUS       sequence-449967-       14332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral Expression of dCas9-KRAB from S. parasanguinis fused to 
            the repressive KRAB domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14332)
  AUTHORS   Butterfield GL, Rohm D, Roberts A, Nethery MA, Rizzo AJ, Morone DJ, 
            Garnier L, Iglesias N, Barrangou R, Gersbach CA
  TITLE     Characterization of diverse Cas9 orthologs for genome and epigenome 
            editing.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Mar 18;122(11):e2417674122. doi: 
            10.1073/pnas.2417674122. Epub 2025 Mar 12.
  PUBMED    40073054
REFERENCE   2  (bases 1 to 14332)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2025-03-18"; volume: "122(11):e2417674122. 
            doi"; pages: " 10.1073/pnas.2417674122. Epub 2025 Mar 12"
COMMENT     Sequence Label: pFUGW hUbC-Spa-dCas9-KRAB-T2A-GFP
FEATURES             Location/Qualifiers
     source          1..14332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        225..423
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     378..398
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             441..621
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    668..793
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1286..1519
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1704..1748
                     /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    2046..2163
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2205..2238)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2262..3473
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3310..3329
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      3502..3511
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3511..3576
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3586..3606
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7048..7068
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7105..7290
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             7297..7350
                     /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(7357..7359,7360..7362,7363..8073)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(7357..7359,7360..7362,7363..8073)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7402..7423)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7663..7682)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8010..8031
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             8083..8139
                     /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"
     protein_bind    complement(8572..8605)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    8630..9218
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8683..8703)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9101..9112)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9221..9237)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9222..9238)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9743..9923
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9949..9966)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9955..10179
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10225..10653
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10312..10331)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10522..10543
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10662..10682)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10667..10996
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10847..10982
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10909..10928
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11044..11091
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11110..11484
                     /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    11614..11735
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11651..11670)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11705..11724
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11784..11800)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11784..11800)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11797..11819)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11808..11824
                     /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(11832..11838,11839..11856,11857..11862))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11877..11898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12015..12032)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12186..12774)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12266..12285)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12945..13736,13737..13805))
                     /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     13568..13587
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13806..13910)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(13985..14004)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(14176..14332,1..223)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
       61 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      121 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      181 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      241 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      301 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      361 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      421 cagcgcgttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
      481 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      541 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      601 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
      661 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
      721 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
      781 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
      841 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
      901 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
      961 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     1021 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     1081 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga
     1141 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     1201 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     1261 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     1321 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     1381 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     1441 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     1501 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     1561 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     1621 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     1681 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     1741 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     1801 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata
     1861 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
     1921 ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc
     1981 gattagtgaa cggatcggca ctgcgtgcgc caattctgca gacaaatggc agtattcatc
     2041 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2101 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2161 tttcgggttt attacaggga cagcagagat ccagtttggt taatataact tcgtatagca
     2221 tacattatac gaagttatta acccgtgtcg gctccagatc tggcctccgc gccgggtttt
     2281 ggcgcctccc gcgggcgccc ccctcctcac ggcgagcgct gccacgtcag acgaagggcg
     2341 cagcgagcgt cctgatcctt ccgcccggac gctcaggaca gcggcccgct gctcataaga
     2401 ctcggcctta gaaccccagt atcagcagaa ggacatttta ggacgggact tgggtgactc
     2461 tagggcactg gttttctttc cagagagcgg aacaggcgag gaaaagtagt cccttctcgg
     2521 cgattctgcg gagggatctc cgtggggcgg tgaacgccga tgattatata aggacgcgcc
     2581 gggtgtggca cagctagttc cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga
     2641 tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc tggccggggc tttcgtggcc
     2701 gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt
     2761 ccgcgagcaa ggttgccctg aactgggggt tggggggagc gcagcaaaat ggcggctgtt
     2821 cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg
     2881 ggggcatggt gggcggcaag aacccaaggt cttgaggcct tcgctaatgc gggaaagctc
     2941 ttattcgggt gagatgggct ggggcaccat ctggggaccc tgacgtgaag tttgtcactg
     3001 actggagaac tcggtttgtc gtctgttgcg ggggcggcag ttatggcggt gccgttgggc
     3061 agtgcacccg tacctttggg agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt
     3121 tggcttataa tgcagggtgg ggccacctgc cggtaggtgt gcggtaggct tttctccgtc
     3181 gcaggacgca gggttcgggc ctagggtagg ctctcctgaa tcgacaggcg ccggacctct
     3241 ggtgagggga gggataagtg aggcgtcagt ttctttggtc ggttttatgt acctatcttc
     3301 ttaagtagct gaagctccgg ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg
     3361 aagtttttta ggcacctttt gaaatgtaat catttgggtc aatatgtaat tttcagtgtt
     3421 agactagtaa attgtccgct aaattctggc cgtttttggc ttttttgtta gacgaagctt
     3481 gggctgcagg tcgactctag agccaccatg gactacaaag accatgacgg tgattataaa
     3541 gatcatgaca tcgattacaa ggatgacgat gacaagcacg ttaaccccaa gaagaagagg
     3601 aaggtgggcc gcggaaccgg tatgaacgga ctggttttgg gtcttgccat cgggatcgct
     3661 agcgttgggg tgggcatcct gaagaaggac ataggagaaa tcattcatac caattctaga
     3721 ctgttttcag ctgccacagc cgactctaat attgaacgac gaggacatcg tggcggcaag
     3781 aggctgacaa gacgaaaaaa acaccgaagc atacgacttc acgatctttt cgaggatttt
     3841 ggactgctga cggacttttc aaaagtttcc atcaacttga atccgtacca gttacgcgta
     3901 cagggtctgg acaaccagct gacaaacgag gagctgttta tcgctcttaa gaatatcgtg
     3961 aagcgccggg ggattagcta cttagacgat gcctctgaag acggcggaac cgtgtcttct
     4021 gattatggga aggctgtcga agaaaataga aaactcttag ccgaacagac tcctgggcag
     4081 atccagctgg acagattcga aaagtacggc caagtccgag gcgacttcaa tgtcgtggag
     4141 aacggtgaga aacgacgtct gattaacgtc tttacaacta gcgcctattc caaggaggcc
     4201 gagaggatac tgaggaagca gcaggagttc aacaagaaaa taacggatga gttcatcgag
     4261 gattacctga ccattcttac tggaaaaaga aaatattacc atggtcctgg aaacgaaaag
     4321 tcccggaccg attacgggcg gtacacaacc aaaaaggacc cagagggcaa atacatcacc
     4381 ctcgataata ttttcgggat cctcatcggt aaatgcactt tttaccccga tgagtatcgc
     4441 gcgtctaaag cttcatatac cgcacaggag ttcaatctgc tcaacgacct gaataacctg
     4501 accgtgccta ccgaaaccaa aaaactgtca gaggagcaga agaagacgat aataaaatac
     4561 gccaaaacgg ctaagaccct tggcgcttct actctgctga agtatatagc caaactgatc
     4621 ggtgcttccg ttgaccagat tcacgggtat agaatcgacc caaataaaaa gcccgaaatg
     4681 cacaccttcg agacgtaccg gaaaatgcaa tccctggaga cgatctcagt ggaggaactg
     4741 cctcgcaaag tgcttgacga actcgcccat attctgacat tgaacactga gcgcgaaggc
     4801 atcgaggagg ctattaatgc caccttgaaa gatacgttta gtcaggacca ggtcctcgaa
     4861 ctcgtgcagt tccgcaaaaa taactcttcc ttattctcaa agggatggca tagtttcagc
     4921 ctgaaactga tgatggaact gattcctgaa ctctatgaga ctagtgaaga gcagatgact
     4981 atactgactc gtctggggaa acagaagtct aaggagacaa gtaaacgaac taagtacatt
     5041 gatgaaaagg agctgacaga agagatttat aatccagtcg tggctaaatc cgtccgtcag
     5101 gctattaaga tcattaacga ggcaacgaaa aagtacggaa tcttcgataa cattgtgatc
     5161 gaaatggccc gtgagaacaa tgaagaagat gctaagaagg agtacatcaa gcggcagaag
     5221 gcaaacttgg atgagaagaa cgccgcaatg gagaaagctg cttttcaata caacggtaag
     5281 aaggaactcc cggataacgt cttccacggc cataaggagc tcgccacaaa aatacggttg
     5341 tggcaccagc agggggaaaa gtgcctctac actggaaaaa atatccctat ctccgacctt
     5401 attcaaaatc agtacaagta tgaaatcgac gccatcctcc cactgtccct cagtttcgac
     5461 gatagcctgt ccaacaaggt cctcgtgctg gctaccgcca atcaggaaaa gggccaaaga
     5521 actccttttc aagctctcga ttcaatggat gacgcctgga gctatcggga gttcaaatcc
     5581 tatgtgaagg attctaaact cttggggaat aaaaagaagg aatacttgtt aacagaggag
     5641 gatatcagta agatcgaggt gaaacagaag tttatcgaac ggaatttagt tgacacaagg
     5701 tactcaagtc gcgtggttct taacgccctt caggacttct acaaggaaca ccagttcgat
     5761 accaccatta gcgtggttag aggacaattt acatcccagc tgcggcggaa atggggactg
     5821 gagaagagcc gagaaaccta ccatcaccac gccgtggacg cgctgattat tgccgccagt
     5881 agccagctcc gcctctggaa aaaacagaac aatcctctta tcagttacac cgaaggccag
     5941 tttgtggatc aggtgaccgg cgagattata tccctttctg acgacgaata caaagagctg
     6001 gttttcaaag ctccatatga ccactttgtt gacacactga agtcaaagaa gtttgaggat
     6061 tctatcctct tttcatacca ggtggattct aagtacaacc ggaagattag cgacgcaacc
     6121 atatatgcaa ctcgtaaagc caaattggac aaggagaata aagagtatac ttacactctg
     6181 ggtaaaatca aagatatcta tgctcttggc acgaaaagcc cttctaaaac cggcttttac
     6241 aagtttctgg acctgtataa taaagacaag tctcagttcc tgatgtttca gaaagatcgc
     6301 aagacctggg acgaggtgat tgaaaagatc attgagcaat accggccttt taaagaatac
     6361 gacgaaaacg gaaaagaagt agacttcaac ccttttgaaa agtaccggat cgaaaatggc
     6421 cccatccgaa aatattccaa aaagggcaat ggcccagaaa taaagagtct caagtattac
     6481 gataacttac tgggtaagtt tgtggatatc acaccttcag agtccaagaa ccccgtagct
     6541 ctgctgtccc tgaacccgtg gaggactgac gtctactaca acaccgaaac atccaagtat
     6601 gagttccttg ggctgaagta tgctgatttg tgcttcgaaa aggggggtgc atatggtatc
     6661 tctgaggtga aatacaataa aataagagag aaagaaggta tcggaaagga gtctgaattc
     6721 aaatttaccc tttataagaa tgacctgatt ctgatcaaag atacagagac aaactgccaa
     6781 cagatttttc gcttctggtc tcgaactgga aaagacaatc ctaaatcatt tgagaagcat
     6841 aaaattgagc ttaaaccgta tgaaaaggct cgtttcgaga agggtgaaga actggaggtg
     6901 ctgggaaagg tgccacctag ttcaaaccaa ctgcagaaaa atatgcaaat tgagaacctc
     6961 tccatctaca aggtgaagac tgatgtgctt ggcaataagc attttatcaa aaaggaagga
     7021 gaagaaccta agctcaaatt taccggtcct aagaaaaagc ggaaagtggc tagcatggat
     7081 gctaagtcac taactgcctg gtcccggaca ctggtgacct tcaaggatgt atttgtggac
     7141 ttcaccaggg aggagtggaa gctgctggac actgctcagc agatcgtgta cagaaatgtg
     7201 atgctggaga actataagaa cctggtttcc ttgggttatc agcttactaa gccagatgtg
     7261 atcctccggt tggagaaggg agaagagccc gctagcgagg gcagaggaag tcttctaaca
     7321 tgcggtgacg tggaggagaa tcccggccct ggaccaatgg tgagcaaggg cgaggagctg
     7381 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     7441 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     7501 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     7561 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     7621 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     7681 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     7741 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     7801 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     7861 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     7921 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     7981 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     8041 gggatcactc tcggcatgga cgagctgtac aagggatccg gcgcaacaaa cttctctctg
     8101 ctgaaacaag ccggagatgt cgaagagaat cctggaccga agaccttcaa catctctcag
     8161 caggatctgg agctggtgga ggtcgccact gagaagatca ccatgctcta tgaggacaac
     8221 aagcaccatg tcggggcggc catcaggacc aagactgggg agatcatctc tgctgtccac
     8281 attgaggcct acattggcag ggtcactgtc tgtgctgaag ccattgccat tgggtctgct
     8341 gtgagcaacg ggcagaagga ctttgacacc attgtggctg tcaggcaccc ctactctgat
     8401 gaggtggaca gatccatcag ggtggtcagc ccctgtggca tgtgcagaga gctcatctct
     8461 gactatgctc ctgactgctt tgtgctcatt gagatgaatg gcaagctggt caaaaccacc
     8521 attgaggaac tcatccccct caagtacacc aggaactaaa gttgataata gataacttcg
     8581 tatagcatac attatacgaa gttatgaatt cgatatcaag cttatcgata atcaacctct
     8641 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     8701 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     8761 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     8821 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     8881 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     8941 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     9001 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     9061 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     9121 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     9181 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga
     9241 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc
     9301 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt
     9361 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
     9421 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca
     9481 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc
     9541 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc
     9601 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc
     9661 ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg
     9721 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct
     9781 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     9841 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     9901 gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     9961 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
    10021 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
    10081 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
    10141 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
    10201 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
    10261 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
    10321 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
    10381 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
    10441 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
    10501 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
    10561 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
    10621 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
    10681 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
    10741 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
    10801 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
    10861 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
    10921 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
    10981 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca
    11041 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
    11101 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc
    11161 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac
    11221 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg
    11281 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag
    11341 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc
    11401 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac
    11461 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc
    11521 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
    11581 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
    11641 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
    11701 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
    11761 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
    11821 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
    11881 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
    11941 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
    12001 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
    12061 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
    12121 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
    12181 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
    12241 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
    12301 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
    12361 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
    12421 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
    12481 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
    12541 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
    12601 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
    12661 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
    12721 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
    12781 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
    12841 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
    12901 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
    12961 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
    13021 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
    13081 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
    13141 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
    13201 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
    13261 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
    13321 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
    13381 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
    13441 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
    13501 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
    13561 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
    13621 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
    13681 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
    13741 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
    13801 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
    13861 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
    13921 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
    13981 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
    14041 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
    14101 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
    14161 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
    14221 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
    14281 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gt
//
