LOCUS       CROPseq-tRFP_(Jiseok)  10322 bp ds-DNA     circular     13-JUL-2022
DEFINITION  .
KEYWORDS    "accession:addgene_86708_317051"
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     Imported from database: AddGene Entry: CROPseq-Guide-Puro URL:
            https://www.addgene.org/browse/sequence/317051/ GENERAL Depositing
            Lab: Christoph Bock Publication: Datlinger et al Nat Methods. 2017
            Jan 18. doi: 10.1038/nmeth.4177. Purpose: Makes CRISPR gRNAs
            detectable by single-cell RNA-seq. The gRNA is expressed as part of
            the puromycin resistance mRNA transcribed by RNA Pol II and in its
            functional form from the hU6 promoter. BACKBONE Backbone
            manufacturer: Feng Zhang Lab Selectable markers: Puromycin Total
            vector size (bp): 10214 Vector backbone: lentiGuide-Puro Vector
            type: Mammalian Expression, Lentiviral, CRISPR CLONING INFORMATION
            5′ sequencing primer: TTGGGCACTGACAATTCCGT Cloning method: Gibson
            Cloning GROWTH IN BACTERIA Bacterial Resistance(s): Ampicillin Copy
            number: High Copy Growth Strain(s): NEB Stable Growth Temperature:
            30°C GENE/INSERT Gene/Insert name: EF1a-Puro-WPRE-hU6-gRNA
            Promoter: hU6 Species: Synthetic gRNA/shRNA sequence: replace
            filler with custom gRNA sequence or library TERMS AND LICENSES
            Academic/Nonprofit Terms:  Industry Terms:  JISEOK <Datlinger 2017>
            Amplification-free cloning of pooled gRNA libraries into the
            CROPseq-Guide-Puro plasmid. Vector backbone was prepared by
            digesting 5 mg of CROPseq-Guide-Puro with 20 units of BsmBI (NEB
            cat. no. R0580L) in a total volume of 25 ml 1× NEB buffer 3.1,
            incubating for 1 h at 55 °C and inactivating the restriction enzyme
            for 20 min at 80 °C. The 8,333 bp fragment was purified using the
            SNAP UV-free gel purification kit (Invitrogen cat. no. K200025).
            gRNAs were synthesized by Sigma Aldrich as 74 base oligonucle-
            otides with 18 and 35 bases of homology to the hU6 promoter and
            guide RNA backbone, respectively. Oligonucleotides were diluted to
            100 mM and pooled in equal amounts. The oligonucle- otide pool was
            further diluted 1:1,000 for a final molarity of 100 nM for assembly
            into the vector backbone. gRNA libraries were cloned by Gibson’s
            isothermal assembly: 11 fmoles (56.7 ng) of CROPseq-Guide-Puro
            backbone and 200 fmoles of pooled ssDNA oligonucleotides (2 ml of
            the 100 nM solution) were combined with 10 ml of NEBuilder HiFi DNA
            assembly master mix (NEB cat. no. E2621S) and water to 20 ml. After
            1 h of incubation at 50 °C, reac- tions were desalted by filter
            dialysis (Merck cat. no. VMWP04700), and 10 ml of the reaction was
            electroporated into 25 ml of Lucigen Endura E. coli cells (Lucigen
            cat. no. 60242-2) using prechilled 1 mm electroporation cuvettes
            (BioRad cat. no. 1652089) in a BioRad GenePulser I machine set to
            25 mF, 200 W, and 1.5 kV. Within sec- onds after the pulse, 1 ml of
            37 °C Recovery Medium (Lucigen) was added and bacteria were grown
            in round-bottom tubes for 1 h at 37 °C while shaking at 180 r.p.m.
            Then, 1 ml of the bacterial culture was plated on a 25 × 25 cm
            bioassay plate containing LB medium (Miller) with 100 μg/ml
            carbenicillin. Plates were incubated at 32 °C for 22 h, then LB
            medium was added and cells were scraped off the plate. Bacterial
            cells were pelleted by 15 min of centrifugation at 5,000 RCF at 4
            °C, and plasmid DNA was extracted with Qiagen’s EndoFree Plasmid
            Mega kit (Qiagen cat. no. 12381). Library cover- age was estimated
            by counting the number of bacterial colonies on a 1:1,000 dilution
            plate. The T-cell receptor library was cloned at 853× coverage.
FEATURES             Location/Qualifiers
     source          1..10322
                     /label="source"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="organism: synthetic DNA construct mol_type: other DNA"
     rep_origin      complement(168..756)
                     /label="ori"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="direction: LEFT note: high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
     primer_bind     complement(248..267)
                     /label="pBR322ori-F"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="note: pBR322 origin, forward primer"
     CDS             complement(927..1787)
                     /label="AmpR"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="gene: bla product: beta-lactamase note: confers resistance to ampicillin, carbenicillin, andrelated antibiotics translation: MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     CDS             complement(927..1787)
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     primer_bind     1550..1569
                     /label="Amp-R"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: Ampicillin resistance gene, reverse primer"
     promoter        complement(1788..1892)
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="gene: bla"
     rep_origin      complement(1918..2373)
                     /label="f1 ori"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="direction: LEFT note: f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
     primer_bind     complement(2055..2076)
                     /label="F1ori-F"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
                     /note="note: F1 origin, forward primer"
     primer_bind     2267..2286
                     /label="F1ori-R"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
                     /note="note: F1 origin, reverse primer"
     primer_bind     2500..2522
                     /label="M13/pUC Forward"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="note: In lacZ gene"
     primer_bind     2514..2531
                     /label="M13 Forward"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="note: In lacZ gene. Also called M13-F20 or M13 (-21) Forward"
     primer_bind     2515..2531
                     /label="M13 fwd"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="note: common sequencing primer, one of multiple similar variants"
     promoter        2541..2559
                     /label="T7 promoter"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="note: promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2541..2560
                     /label="T7"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="note: T7 promoter, forward primer"
     rep_origin      2580..2715
                     /label="SV40 ori"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
                     /note="note: SV40 origin of replication"
     primer_bind     complement(2634..2653)
                     /label="SV40pro-F"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
                     /note="note: SV40 promoter/origin, forward primer"
     polyA_signal    2755..2876
                     /label="SV40 poly(A) signal"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: SV40 polyadenylation signal"
     primer_bind     complement(2766..2785)
                     /label="EBV-rev"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="note: SV40 polyA terminator, reverse primer"
     primer_bind     2820..2839
                     /label="SV40pA-R"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /note="note: SV40 polyA, reverse primer"
     LTR             2954..3134
                     /label="5' LTR (truncated)"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="note: truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3152..3178
                     /label="i7 primer"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     misc_RNA        complement(3187..3262)
                     /label="gRNA scaffold"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /note="note: guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system"
     misc_feature    complement(3228..3262)
                     /label="gRNA 3' postfix"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     misc_feature    complement(5143..5161)
                     /label="gRNA 5' prefix"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
     misc_feature    5143..5165
                     /label="i5 primer"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     misc_feature    5144..3258
                     /label="BsmB1 cut fragment"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
     promoter        complement(5152..5392)
                     /label="U6 promoter"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
                     /note="note: RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(5202..5221)
                     /label="LKO.1 5'"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /note="note: Human U6 promoter, forward primer"
     primer_bind     complement(5372..5392)
                     /label="hU6-F"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: Human U6 promoter, forward primer"
     misc_feature    5548..6136
                     /label="WPRE"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
                     /note="note: woodchuck hepatitis virus posttranscriptional regulatory element"
     CDS             5654..5665
                     /label="Factor Xa site"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
                     /note="product: Factor Xa recognition and cleavage site translation: IEGR"
     CDS             5654..5665
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="IEGR"
     primer_bind     6063..6083
                     /label="WPRE-R"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
                     /note="note: WPRE, reverse primer"
     misc_feature    complement(6152..6865)
                     /label="tRFP"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     misc_feature    6867..6146
                     /label="BsiW1 Mlu1 cut"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     promoter        complement(6872..8050)
                     /label="EF-1-alpha promoter"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="note: strong constitutive promoter for human elongation factor EF-1-alpha"
     intron          6881..7819
                     /label="EF-1-alpha intron A"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="note: intron upstream of the start codon of human EF-1-alpha"
     primer_bind     complement(6904..6924)
                     /label="EF1a-F"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="note: Human elongation factor-1a promoter, forward primer"
     misc_feature    8189..8306
                     /label="cPPT/CTS"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
                     /note="note: central polypurine tract and central termination sequence of HIV-1"
     CDS             complement(8487..8531)
                     /label="gp41 peptide"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="product: antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013) note: recognized by the 2H10 single-chain llama nanobody translation: KNEQELLELDKWASL"
     CDS             complement(8487..8531)
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /translation="KNEQELLELDKWASL"
     misc_feature    8716..8949
                     /label="RRE"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="note: The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm."
     misc_feature    9442..9567
                     /label="HIV-1 Psi"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: packaging signal of human immunodeficiency virus type 1"
     LTR             9614..9794
                     /label="5' LTR (truncated)"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="note: truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(9795..10021)
                     /label="RSV promoter"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="note: Rous sarcoma virus enhancer/promoter"
     promoter        complement(10049..10067)
                     /label="T3 promoter"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
                     /note="note: promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10049..10069)
                     /label="T3"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="note: T3 promoter, forward primer"
     primer_bind     complement(10088..10104)
                     /label="M13 Reverse"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
                     /note="note: In lacZ gene. Also called M13-rev"
     primer_bind     complement(10088..10104)
                     /label="M13 rev"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="note: common sequencing primer, one of multiple similar variants"
     primer_bind     complement(10101..10123)
                     /label="M13/pUC Reverse"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="note: In lacZ gene"
     protein_bind    10112..10128
                     /label="lac operator"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="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(10136..10166)
                     /label="lac promoter"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /note="note: promoter for the E. coli lac operon"
     protein_bind    10181..10202
                     /label="CAP binding site"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="bound_moiety: E. coli catabolite activator protein note: CAP binding activates transcription in the presence of cAMP."
     primer_bind     complement(10319..14)
                     /label="L4440"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="note: L4440 vector, forward primer"
ORIGIN
        1 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
       61 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      121 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      181 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      241 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      301 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      361 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      421 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      481 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      541 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      601 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      661 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      721 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      781 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
      841 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
      901 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
      961 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1021 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1081 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1141 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1201 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1261 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     1321 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     1381 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1441 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1501 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1561 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     1621 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     1681 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     1741 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     1801 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     1861 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     1921 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     1981 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     2041 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     2101 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     2161 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2221 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2281 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2341 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     2401 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     2461 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     2521 cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt acgtccctag
     2581 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc
     2641 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     2701 agttaggggc gggatgggcg gagttagggg cgggatagct agagccagac atgataagat
     2761 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     2821 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttcctc
     2881 tcactctctg atattcattt ctttgcaagt tataaatact gaataataag atgacatgaa
     2941 ctactatacg tactgctaga gattttccac actgactaaa agggtctgag ggatctctag
     3001 ttaccagagt cacacaacag acgggcacac actacttgaa gcactcaagg caagctttat
     3061 tgaggcttaa gcagtgggtt ccctagttag ccagagagct cccaggctca gatctggtct
     3121 aaccagagag acccagtaca agcaaaaagc agtgtctcaa gatctagtta cgccaagctt
     3181 aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac
     3241 ttgctatttc tagctctaaa acagagacgt acaaaaaaga gcaagaagct aaaaaagatt
     3301 taaaaattat ttttagcgca gttaatggaa caggaactaa atttacccca aaaatattac
     3361 gtgaatcagg atataacgtt attgaggttg aagagcatgc atttgaagat gaaacattta
     3421 aaaatgttgt aaatccaaat ccagaatttg atcctgcatg aaaaataccg cttgaatatg
     3481 gtattaaaca tgatgcagat attattatta tgaatgaccc agatgctgac agatttggaa
     3541 tggcaataaa acatgatggt cattttgtaa gattagatgg aaatcaaaca ggaccaattt
     3601 taattgattg aaaattatca aatctaaaac gcttaaatag cattccaaaa aatccggctc
     3661 tatattcaag ttttgtaaca agtgatttgg gtgatagaat cgctcatgaa aaatatggag
     3721 ttaatattgt aaaaacttta actggattta aatgaatggg tagagaaatt gctaaagaag
     3781 aagataacgg attaaatttt gtttttgctt atgaagaaag ttatggatat gtaattgatg
     3841 actcagctag agataaagat ggaatacaag cttctatatt aatagcagag gctgcttgat
     3901 tttataaaaa acaaaataaa acattagtag actatttaga agatttattt aaagaaatgg
     3961 gtgcatatta cactttcact ttaaacttga attttaaacc agaagaaaag aaattaaaaa
     4021 ttgaaccatt aatgaaatca ttgagagcaa cacccttaac tcaaattgct ggacttaaag
     4081 ttgttaatgt tgaagactac atcgatggaa tgtataatat gccaggacaa gacttactaa
     4141 aattttattt agaagataag tcatgatttg ctgttcgccc aagtggaact gaacctaaac
     4201 taaaaattta ttttataggt gttggtgaat ctgttcaaaa cgctaaagtt aaagtagacg
     4261 aaattattaa agaattaaaa ttaaaaatga atatatagga gaaaaaatga aactaaacaa
     4321 atatatagat cacacattat taaaacaaga tgctacgaaa gctgaaatta aacaattatg
     4381 tgatgaagca attgaatttg attttgcaac agtttgtgtt aattcatatt gaacaagcta
     4441 ttgtaaagaa ttattaaaag gcacaaatgt aggaataaca aatgttgtag gttttcctct
     4501 aggtgcatgc acaacagcta caaaagcatt cgaagtttct gaagcaatta aagatggtgc
     4561 aacagaaatt gatatggtat taaatattgg tgcattaaaa gacaaaaatt atgaattagt
     4621 tttagaagac atgaaagctg taaaaaaagc agctggatca catgttgtta aatgtattat
     4681 ggaaaattgt ttattaacaa aagaagaaat catgaaagct tgtgaaatag ctgttgaagc
     4741 tggattagaa tttgttaaaa catcaacagg attttcaaaa tcaggtgcaa catttgaaga
     4801 tgttaaacta atgaagtcag ttgttaaaga caatgcttta gttaaagcag ctggtggagt
     4861 tagaacattt gaagatgctc aaaaaatgat tgaagcagga gctgaccgct taggaacaag
     4921 tggtggagta gctattatta aaggtgaaga aaacaacgcg agttactaaa actagcgttt
     4981 ttttattttg ctcattttta ttaaaagttt gcaaaaagga acataaaaat tctaattatt
     5041 gatactaaag ttattaaaaa gaagattttg gttgatttta taaaggtcat agaatataat
     5101 attttagcat gtgtattttg tgtgctcatt tacaaccgtc tccggtgttt cgtcctttcc
     5161 acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt
     5221 ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc
     5281 acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt atctctctaa
     5341 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tctctgggtc
     5401 ccctcggggt tgggaggtgg gtctgaaacg ataatggtga aatcttgtct tcgttgggag
     5461 tgaattagcc cttccagtcc ccccttttct tttaaaaagt ggctaagatc tacagctgcc
     5521 ttgtaagtca ttggtcttaa agtcgacgcg gggaggcggc ccaaagggag atccgactcg
     5581 tctgagggcg aaggcgaaga cgcggaagag gccgcagagc cggcagcagg ccgcgggaag
     5641 gaaggtccgc tggattgagg gccgaaggga cgtagcagaa ggacgtcccg cgcagaatcc
     5701 aggtggcaac acaggcgagc agccaaggaa aggacgatga tttccccgac aacaccacgg
     5761 aattgtcagt gcccaacagc cgagcccctg tccagcagcg ggcaaggcag gcggcgatga
     5821 gttccgccgt ggcaataggg agggggaaag cgaaagtccc ggaaaggagc tgacaggtgg
     5881 tggcaatgcc ccaaccagtg ggggttgcgt cagcaaacac agtgcacacc acgccacgtt
     5941 gcctgacaac gggccacaac tcctcataaa gagacagcaa ccaggattta tacaaggagg
     6001 agaaaatgaa agccatacgg gaagcaatag catgatacaa aggcattaaa gcagcgtatc
     6061 cacatagcgt aaaaggagca acatagttaa gaataccagt caatctttca caaattttgt
     6121 aatccagagg ttgattgtcg acttaacgcg tTCAATTAAG TTTGTGCCCC AGTTTGCTAG
     6181 GGAGGTCGCA GTATCTGGCC ACAGCCACCT CGTGCTGCTC GACGTAGGTC TCTTTGTCGG
     6241 CCTCCTTGAT TCTTTCCAGT CTGTGGTCCA CATAGTAGAC GCCGGGCATC TTGAGGTTCT
     6301 TAGCGGGTTT CTTGGATCTG TATGTGGTCT TGAAGTTGCA GATCAGGTGG CCCCCGCCCA
     6361 CGAGCTTCAG GGCCATGTCG CTTCTGCCTT CCAGGCCGCC GTCAGCGGGG TACAGCATCT
     6421 CGGTGTTGGC CTCCCAGCCG AGTGTTTTCT TCTGCATCAC AGGGCCGTTG GATGGGAAGT
     6481 TCACCCCTCT GATCTTGACG TTGTAGATGA GGCAGCCGTC CTGGAGGCTG GTGTCCTGGG
     6541 TAGCGGTCAG CACGCCCCCG TCTTCGTATG TGGTGACTCT CTCCCATGTG AAGCCCTCAG
     6601 GGAAGGACTG CTTAAAGAAG TCGGGGATGC CCTGGGTGTG GTTGATGAAG GTTCTGCTGC
     6661 CGTACATGAA GCTGGTAGCC AGGATGTCGA AGGCGAAGGG GAGAGGGCCG CCCTCGACCA
     6721 CCTTGATTCT CATGGTCTGG GTGCCCTCGT AGGGCTTGCC TTCGCCCTCG GATGTGCACT
     6781 TGAAGTGGTG GTTGTTCACG GTGCCCTCCA TATACAGCTT CATGTGCATG TTCTCCTTAA
     6841 TCAGCTCTTC GCCCTTAGAC ACCATcgtac gtcacgacac ctgaaatgga agaaaaaaac
     6901 tttgaaccac tgtctgaggc ttgagaatga accaagatcc aaactcaaaa agggcaaatt
     6961 ccaaggagaa ttacatcaag tgccaagctg gcctaacttc agtctccacc cactcagtgt
     7021 ggggaaactc catcgcataa aacccctccc cccaacctaa agacgacgta ctccaaaagc
     7081 tcgagaacta atcgaggtgc ctggacggcg cccggtactc cgtggagtca catgaagcga
     7141 cggctgagga cggaaaggcc cttttccttt gtgtgggtga ctcacccgcc cgctctcccg
     7201 agcgccgcgt cctccatttt gagctccctg cagcagggcc gggaagcggc catctttccg
     7261 ctcacgcaac tggtgccgac cgggccagcc ttgccgccca gggcggggcg atacacggcg
     7321 gcgcgaggcc aggcaccaga gcaggccggc cagcttgaga ctacccccgt ccgattctcg
     7381 gtggccgcgc tcgcaggccc cgcctcgccg aacatgtgcg ctgggacgca cgggccccgt
     7441 cgccgcccgc ggccccaaaa accgaaatac cagtgtgcag atcttggccc gcatttacaa
     7501 gactatcttg ccagaaaaaa agcgtcgcag caggtcatca aaaattttaa atggctagag
     7561 acttatcgaa agcagcgaga caggcgcgaa ggtgccacca gattcgcacg cggcggcccc
     7621 agcgcccagg ccaggcctca actcaagcac gaggcgaagg ggctccttaa gcgcaaggcc
     7681 tcgaactctc ccacccactt ccaacccgaa gctcgggatc aagaatcacg tactgcagcc
     7741 aggtggaagt aattcaaggc acgcaagggc cataacccgt aaagaggcca ggcccgcggg
     7801 aaccacacac ggcacttacc tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca
     7861 cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg cggagccagt
     7921 acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg gatcaattgc
     7981 cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc cactgacggg
     8041 caccggagcc aattcccact cctttcaaga cctagaaggt ccattagctg caaagattcc
     8101 tctctgttta aaactttatc catctttgca cccgggcccc ctcgagccgg cgccaaagtg
     8161 gatctctgct gtccctgtaa taaacccgaa aattttgaat ttttgtaatt tgtttttgta
     8221 attctttagt ttgtatgtct gttgctatta tgtctactat tctttcccct gcactgtacc
     8281 ccccaatccc cccttttctt ttaaaattgt ggatgaatac tgccatttgt ctcaagattc
     8341 tgaaacgata atggtgaata tccctgccta actctattca ctatagaaag tacagcaaaa
     8401 actattctta aacctaccaa gcctcctact atcattatga ataattttat ataccacagc
     8461 caatttgtta tgttaaacca attccacaaa cttgcccatt tatctaattc caataattct
     8521 tgttcattct tttcttgctg gttttgcgat tcttcaatta aggagtgtat taagcttgtg
     8581 taattgttaa tttctctgtc ccactccatc caggtcgtgt gattccaaat ctgttccaga
     8641 gatttattac tccaactagc attccaaggc acagcagtgg tgcaaatgag ttttccagag
     8701 caaccccaaa tccccaggag ctgttgatcc tttaggtatc tttccacagc caggattctt
     8761 gcctggagct gcttgatgcc ccagactgtg agttgcaaca gatgctgttg cgcctcaata
     8821 gccctcagca aattgttctg ctgctgcact ataccagaca ataattgtct ggcctgtacc
     8881 gtcagcgtca ttgacgctgc gcccatagtg cttcctgctg ctcccaagaa cccaaggaac
     8941 aaagctccta ttcccactgc tcttttttct ctctgcacca ctcttctctt tgccttggtg
     9001 ggtgctactc ctaatggttc aatttttact actttatatt tatataattc acttctccaa
     9061 ttgtccctca tatctcctcc tccaggtctg aagatcagcg gccgcttgct gtgcggtggt
     9121 cttacttttg ttttgctctt cctctatctt gtctaaagct tccttggtgt cttttatctc
     9181 tatcctttga tgcacacaat agagggttgc tactgtatta tataatgatc taagttcttc
     9241 tgatcctgtc tgaagggatg gttgtagctg tcccagtatt tgtctacagc cttctgatgt
     9301 ttctaacagg ccaggattaa ctgcgaatcg ttctagctcc ctgcttgccc atactatatg
     9361 ttttaattta tattttttct ttccccctgg ccttaaccga attttttccc atcgcgatct
     9421 aattctcccc cgcttaatac tgacgctctc gcacccatct ctctccttct agcctccgct
     9481 agtcaaaatt tttggcgtac tcaccagtcg ccgcccctcg cctcttgccg tgcgcgcttc
     9541 agcaagccga gtcctgcgtc gagagagctc ctctggtttc cctttcgctt tcaagtccct
     9601 gttcgggcgc cactgctaga gattttccac actgactaaa agggtctgag ggatctctag
     9661 ttaccagagt cacacaacag acgggcacac actacttgaa gcactcaagg caagctttat
     9721 tgaggcttaa gcagtgggtt ccctagttag ccagagagct cccaggctca gatctggtct
     9781 aaccagagag acccgtttat gtatcgagct aggcacttaa atacaatatc tctgcaatgc
     9841 ggcaattcag tggttcgtcc aatccatgtc agacccgtct gttgccttcc taataaggca
     9901 cgatcgtacc accttacttc caccaatcgg catgcacggt gctttttctc tccttgtaag
     9961 gcatgttgct aactcatcgt taccatgttg caagactaca agagtattgc ataagactac
    10021 attaagcttg cagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
    10081 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
    10141 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
    10201 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
    10261 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
    10321 cg
//