LOCUS       sequence-381970-       13729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA and dSaCas9-KRAB from lentiviral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13729)
  AUTHORS   McCutcheon SR, Swartz AM, Brown MC, Barrera A, McRoberts Amador C, 
            Siklenka K, Humayun L, Isaacs JM, Reddy TE, Nair S, Antonia S, 
            Gersbach CA
  TITLE     Orthogonal CRISPR screens to identify transcriptional and epigenetic
            regulators of human CD8 T cell function.
  JOURNAL   bioRxiv. 2023 May 1:2023.05.01.538906. doi: 
            10.1101/2023.05.01.538906. Preprint.
  PUBMED    37205457
REFERENCE   2  (bases 1 to 13729)
  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: "bioRxiv. 
            2023 May 1:2023.05.01.538906. doi: 10.1101/2023.05.01.538906. 
            Preprint."
COMMENT     Sequence Label: pLV hU6-gRNA hUbC-dSaCas9-KRAB-T2A-Thy1.1
FEATURES             Location/Qualifiers
     source          1..13729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(71..90)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        262..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        643..841
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             859..1039
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1086..1211
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1704..1937
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2122..2166
                     /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    2464..2581
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        complement(2664..2739)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     primer_bind     complement(2814..2833)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3026..4237
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4074..4093
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4275..4301
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4308..4328
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7509..7556
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7590..7784
                     /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="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             7851..7871
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7878..7931
                     /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             7932..8420
                     /codon_start=1
                     /product="mouse GPI-anchored cell surface glycoprotein of 
                     the immunoglobulin superfamily"
                     /label=Thy1.1
                     /note="This allele of Thy1 is also known as CD90.1."
                     /translation="MNPAISVALLLSVLQVSRGQKVTSLTACLVNQNLRLDCRHENNTK
                     DNSIQHEFSLTREKRKHVLSGTLGIPEHTYRSRVTLSNQPYIKVLTLANFTTKDEGDYF
                     CELRVSGANPMSSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL"
     misc_feature    8445..9033
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8498..8518)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8916..8927)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9036..9052)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9037..9053)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9558..9738
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9764..9781)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9770..9994
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10040..10468
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10127..10146)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10337..10358
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10477..10497)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10482..10811
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10662..10797
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10724..10743
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10859..10906
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10925..11299
                     /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    11429..11550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11466..11485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11520..11539
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11599..11615)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11599..11615)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11612..11634)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11623..11639
                     /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(11647..11653,11654..11671,11672..11677))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11692..11713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11830..11847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12001..12589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12081..12100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12760..13551,13552..13620))
                     /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     13383..13402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13621..13725)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
       61 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
      121 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
      181 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      241 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      781 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gcgcgttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
      901 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
      961 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     1021 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     1081 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     1141 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     1201 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     1261 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     1321 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     1381 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     1441 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     1501 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc
     1561 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     1621 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     1681 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     1741 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     1801 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     1861 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     1921 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     1981 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     2041 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     2101 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     2161 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     2221 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     2281 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     2341 gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga
     2401 ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag tattcatcca
     2461 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2521 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2581 tcgggtttat tacagggaca gcagagatcc agtttggtta atgaacaaaa gctggagctc
     2641 caccgcgggc ggccgcaaaa aaatctcgcc aacaagttga cgagataaac acggcatttt
     2701 gccttgtttt agtagattct gtttccagag tactaaaaca gagacgtccg tctccggtgt
     2761 ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa
     2821 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt
     2881 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattccaa
     2941 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc
     3001 attaacccgt gtcggctcca gatctggcct ccgcgccggg ttttggcgcc tcccgcgggc
     3061 gcccccctcc tcacggcgag cgctgccacg tcagacgaag ggcgcagcga gcgtcctgat
     3121 ccttccgccc ggacgctcag gacagcggcc cgctgctcat aagactcggc cttagaaccc
     3181 cagtatcagc agaaggacat tttaggacgg gacttgggtg actctagggc actggttttc
     3241 tttccagaga gcggaacagg cgaggaaaag tagtcccttc tcggcgattc tgcggaggga
     3301 tctccgtggg gcggtgaacg ccgatgatta tataaggacg cgccgggtgt ggcacagcta
     3361 gttccgtcgc agccgggatt tgggtcgcgg ttcttgtttg tggatcgctg tgatcgtcac
     3421 ttggtgagta gcgggctgct gggctggccg gggctttcgt ggccgccggg ccgctcggtg
     3481 ggacggaagc gtgtggagag accgccaagg gctgtagtct gggtccgcga gcaaggttgc
     3541 cctgaactgg gggttggggg gagcgcagca aaatggcggc tgttcccgag tcttgaatgg
     3601 aagacgcttg tgaggcgggc tgtgaggtcg ttgaaacaag gtggggggca tggtgggcgg
     3661 caagaaccca aggtcttgag gccttcgcta atgcgggaaa gctcttattc gggtgagatg
     3721 ggctggggca ccatctgggg accctgacgt gaagtttgtc actgactgga gaactcggtt
     3781 tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt gggcagtgca cccgtacctt
     3841 tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagg
     3901 gtggggccac ctgccggtag gtgtgcggta ggcttttctc cgtcgcagga cgcagggttc
     3961 gggcctaggg taggctctcc tgaatcgaca ggcgccggac ctctggtgag gggagggata
     4021 agtgaggcgt cagtttcttt ggtcggtttt atgtacctat cttcttaagt agctgaagct
     4081 ccggttttga actatgcgct cggggttggc gagtgtgttt tgtgaagttt tttaggcacc
     4141 ttttgaaatg taatcatttg ggtcaatatg taattttcag tgttagacta gtaaattgtc
     4201 cgctaaattc tggccgtttt tggctttttt gttagacgaa gcttgggctg caggtcgact
     4261 ctagagccac catgtaccca tacgatgttc cagattacgc tatggcccca aagaagaagc
     4321 ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa ctacatcctg ggcctggcca
     4381 tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg
     4441 ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggtagg cggagcaaga
     4501 gaggcgccag aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc
     4561 tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg
     4621 ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc
     4681 acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg
     4741 agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg
     4801 ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat
     4861 tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc
     4921 accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct
     4981 actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg
     5041 agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct
     5101 acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg
     5161 agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga
     5221 agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta
     5281 agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg
     5341 acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga
     5401 ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc
     5461 tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg
     5521 gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca
     5581 acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt
     5641 cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga
     5701 agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc
     5761 ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga
     5821 tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga
     5881 ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg
     5941 aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct
     6001 tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca
     6061 acaaggtgct cgtgaagcag gaagaagcca gcaagaaggg caaccggacc ccattccagt
     6121 acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc
     6181 tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg
     6241 acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat
     6301 acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg
     6361 tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta
     6421 agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg
     6481 ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc
     6541 agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca
     6601 aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt
     6661 acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca
     6721 cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca
     6781 aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc
     6841 accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga
     6901 agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa
     6961 aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc
     7021 tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaagc
     7081 cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc gtgaagaatc
     7141 tggatgtgat caaaaaagaa aactactacg aagtgaatag caagtgctat gaggaagcta
     7201 agaagctgaa gaagatcagc aaccaggccg agtttatcgc ctccttctac aacaacgatc
     7261 tgatcaagat caacggcgag ctgtatagag tgatcggcgt gaacaacgac ctgctgaacc
     7321 ggatcgaagt gaacatgatc gacatcacct accgcgagta cctggaaaac atgaacgaca
     7381 agaggccccc caggatcatt aagacaatcg cctccaagac ccagagcatt aagaagtaca
     7441 gcacagacat tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct cagatcatca
     7501 aaaagggcaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat
     7561 ccgatgctaa gtcactgact gcctggtccc ggacactggt gaccttcaag gatgtgtttg
     7621 tggacttcac cagggaggag tggaagctgc tggacactgc tcagcagatc ctgtacagaa
     7681 atgtgatgct ggagaactat aagaacctgg tttccttggg ttatcagctt actaagccag
     7741 atgtgatcct ccggttggag aagggagaag agccctggct ggtggagaga gaaattcacc
     7801 aagagaccca tcctgattca gagactgcat ttgaaatcaa atcatcagtt ccgaaaaaga
     7861 aacgcaaagt tgctagcgag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga
     7921 atcccggccc tatgaaccca gccatcagcg tcgctctcct gctctcagtc ttgcaggtgt
     7981 cccgagggca gaaggtgacc agcctgacag cctgcctggt gaaccaaaac cttcgcctgg
     8041 actgccgcca tgagaataac accaaggata actccatcca gcatgagttc agcctgaccc
     8101 gagagaagag gaagcacgtg ctctcaggca cccttgggat acccgagcac acgtaccgct
     8161 cccgcgtcac cctctccaac cagccctata tcaaggtcct taccctagcc aacttcacca
     8221 ccaaggatga gggcgactac ttttgtgagc ttcgcgtttc gggcgcgaat cccatgagct
     8281 ccaataaaag tatcagtgtg tatagagaca agctggtcaa gtgtggcggc ataagcctgc
     8341 tggttcagaa cacatcctgg atgctgctgc tgctgctttc cctctccctc ctccaagccc
     8401 tggacttcat ttctctgtga gaattcgata tcaagcttat cgataatcaa cctctggatt
     8461 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     8521 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     8581 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     8641 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     8701 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     8761 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     8821 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     8881 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     8941 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     9001 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgagacctag
     9061 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc
     9121 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac
     9181 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     9241 aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac
     9301 aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga
     9361 cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg
     9421 aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga
     9481 gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc
     9541 tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     9601 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     9661 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     9721 tgtggaaaat ctctagcagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     9781 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     9841 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     9901 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     9961 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
    10021 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
    10081 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
    10141 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
    10201 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
    10261 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
    10321 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
    10381 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
    10441 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
    10501 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
    10561 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
    10621 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
    10681 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
    10741 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
    10801 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt
    10861 tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta
    10921 aaccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc
    10981 ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc
    11041 cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc
    11101 ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc
    11161 ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga
    11221 gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt
    11281 ggccgaggag caggactgac acgtgctacg agatttcgat tccaccgccg ccttctatga
    11341 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
    11401 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
    11461 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
    11521 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
    11581 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
    11641 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
    11701 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
    11761 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
    11821 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
    11881 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
    11941 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
    12001 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
    12061 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
    12121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
    12181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
    12241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
    12301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
    12361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
    12421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
    12481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
    12541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
    12601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
    12661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
    12721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
    12781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
    12841 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
    12901 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
    12961 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
    13021 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
    13081 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
    13141 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
    13201 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
    13261 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
    13321 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
    13381 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
    13441 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
    13501 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
    13561 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
    13621 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
    13681 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcaca
//
