LOCUS       sequence-324511-       13466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of sgASCL1 paired with a second S. pyogenes 
            sgRNA with a GFP-P2A-PuroR selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13466)
  AUTHORS   Black JB, McCutcheon SR, Dube S, Barrera A, Klann TS, Rice GA, Adkar
            SS, Soderling SH, Reddy TE, Gersbach CA
  TITLE     Master Regulators and Cofactors of Human Neuronal Cell Fate 
            Specification Identified by CRISPR Gene Activation Screens.
  JOURNAL   Cell Rep. 2020 Dec 1;33(9):108460. doi: 
            10.1016/j.celrep.2020.108460.
  PUBMED    33264623
REFERENCE   2  (bases 1 to 13466)
  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: "Cell Rep.";
            date: "2020-12-1"; volume: "33(9):108460. doi"; pages: " 
            10.1016/j.celrep.2020.108460"
COMMENT     Sequence Label: pLV_mU6-sgASCL1_hU6-sgRNA_hUbC-PuroR-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..13466
                     /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"
     primer_bind     2208..2224
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2243..2263
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2245..2263
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(4264..4512)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4322..4341)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4492..4512)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4526..4545)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4527..4545)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4555..4572)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4555..4571)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(4793..5106)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(4854..4874)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     6119..6138
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             join(6311..6313,6314..6316,6317..7027)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6311..6313,6314..6316,6317..7027)
                     /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(6356..6377)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6617..6636)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6964..6985
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7037..7093
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7094..7693
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7094..7113)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7590..7610
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(7706..7739)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    7764..8352
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7817..7837)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8235..8246)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8355..8371)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8356..8372)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8877..9057
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9083..9100)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9089..9313
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9359..9787
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9446..9465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9656..9677
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9796..9816)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9801..10130
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9981..10116
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10043..10062
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10178..10225
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10244..10618
                     /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    10748..10869
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10785..10804)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10839..10858
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10918..10934)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10931..10953)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10942..10958
                     /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(10966..10972,10973..10990,10991..10996))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11011..11032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11149..11166)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11320..11908)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11400..11419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12079..12870,12871..12939))
                     /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     12702..12721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12940..13044)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(13119..13138)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(13310..13466,1..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 taatacacag gaaacagcta
     2221 tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct
     2281 ccaccgcgga aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc
     2341 ttatttaaac ttgctatgct gtttccagca tagctcttaa acagagacgt acaaaaaaga
     2401 gcaagaagct aaaaaagatt taaaaattat ttttagcgca gttaatggaa caggaactaa
     2461 atttacccca aaaatattac gtgaatcagg atataacgtt attgaggttg aagagcatgc
     2521 atttgaagat gaaacattta aaaatgttgt aaatccaaat ccagaatttg atcctgcatg
     2581 aaaaataccg cttgaatatg gtattaaaca tgatgcagat attattatta tgaatgaccc
     2641 agatgctgac agatttggaa tggcaataaa acatgatggt cattttgtaa gattagatgg
     2701 aaatcaaaca ggaccaattt taattgattg aaaattatca aatctaaaac gcttaaatag
     2761 cattccaaaa aatccggctc tatattcaag ttttgtaaca agtgatttgg gtgatagaat
     2821 cgctcatgaa aaatatggag ttaatattgt aaaaacttta actggattta aatgaatggg
     2881 tagagaaatt gctaaagaag aagataacgg attaaatttt gtttttgctt atgaagaaag
     2941 ttatggatat gtaattgatg actcagctag agataaagat ggaatacaag cttctatatt
     3001 aatagcagag gctgcttgat tttataaaaa acaaaataaa acattagtag actatttaga
     3061 agatttattt aaagaaatgg gtgcatatta cactttcact ttaaacttga attttaaacc
     3121 agaagaaaag aaattaaaaa ttgaaccatt aatgaaatca ttgagagcaa cacccttaac
     3181 tcaaattgct ggacttaaag ttgttaatgt tgaagactac atcgatggaa tgtataatat
     3241 gccaggacaa gacttactaa aattttattt agaagataag tcatgatttg ctgttcgccc
     3301 aagtggaact gaacctaaac taaaaattta ttttataggt gttggtgaat ctgttcaaaa
     3361 cgctaaagtt aaagtagacg aaattattaa agaattaaaa ttaaaaatga atatatagga
     3421 gaaaaaatga aactaaacaa atatatagat cacacattat taaaacaaga tgctacgaaa
     3481 gctgaaatta aacaattatg tgatgaagca attgaatttg attttgcaac agtttgtgtt
     3541 aattcatatt gaacaagcta ttgtaaagaa ttattaaaag gcacaaatgt aggaataaca
     3601 aatgttgtag gttttcctct aggtgcatgc acaacagcta caaaagcatt cgaagtttct
     3661 gaagcaatta aagatggtgc aacagaaatt gatatggtat taaatattgg tgcattaaaa
     3721 gacaaaaatt atgaattagt tttagaagac atgaaagctg taaaaaaagc agctggatca
     3781 catgttgtta aatgtattat ggaaaattgt ttattaacaa aagaagaaat catgaaagct
     3841 tgtgaaatag ctgttgaagc tggattagaa tttgttaaaa catcaacagg attttcaaaa
     3901 tcaggtgcaa catttgaaga tgttaaacta atgaagtcag ttgttaaaga caatgcttta
     3961 gttaaagcag ctggtggagt tagaacattt gaagatgctc aaaaaatgat tgaagcagga
     4021 gctgaccgct taggaacaag tggtggagta gctattatta aaggtgaaga aaacaacgcg
     4081 agttactaaa actagcgttt ttttattttg ctcattttta ttaaaagttt gcaaaaagga
     4141 acataaaaat tctaattatt gatactaaag ttattaaaaa gaagattttg gttgatttta
     4201 taaaggtcat agaatataat attttagcat gtgtattttg tgtgctcatt tacaaccgtc
     4261 tccggtgttt cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt
     4321 tacggtaagc atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag
     4381 aaattattac tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt
     4441 aattccaatt atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg
     4501 aaataggccc tcggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     4561 cgtcgtttta caacattaac ccgtgtcggc tccagatctg gcctccgcgc cgggttttgg
     4621 cgcctcccgc gggcgccccc ctcctcacgg cgagctgaat ctgggcgact cagtacaaaa
     4681 aaagcagccg actcggctgc cactttttca agttgtgtac ggactagcct tatttgaact
     4741 tgctatgcag ctttctgctt agctctcaaa ctaaactccc cctcctcctc caaacaaggc
     4801 ttttctccaa gggatattta tagtctcaaa acacacaatt actttacagt tagggtgagt
     4861 ttccttttgt gctgtttttt aaaataataa tttagtattt gtatctctta tagaaatcca
     4921 agcctatcat gtaaaatgta gctagtatta aaaagaacag attatctgtc ttttatcgca
     4981 cattaagcct ctatagttac taggaaatat tatatgcaaa ttaaccgggg caggggagta
     5041 gccgagcttc tcccacaagt ctgtgcgagg gggccggcgc gggcctagag atggcggcgt
     5101 cggatctatc tgagaccggg ctaacggctg ccacgtcaga cgaagggcgc agcgagcgtc
     5161 ctgatccttc cgcccggacg ctcaggacag cggcccgctg ctcataagac tcggccttag
     5221 aaccccagta tcagcagaag gacattttag gacgggactt gggtgactct agggcactgg
     5281 ttttctttcc agagagcgga acaggcgagg aaaagtagtc ccttctcggc gattctgcgg
     5341 agggatctcc gtggggcggt gaacgccgat gattatataa ggacgcgccg ggtgtggcac
     5401 agctagttcc gtcgcagccg ggatttgggt cgcggttctt gtttgtggat cgctgtgatc
     5461 gtcacttggt gagtagcggg ctgctgggct ggccggggct ttcgtggccg ccgggccgct
     5521 cggtgggacg gaagcgtgtg gagagaccgc caagggctgt agtctgggtc cgcgagcaag
     5581 gttgccctga actgggggtt ggggggagcg cagcaaaatg gcggctgttc ccgagtcttg
     5641 aatggaagac gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg gggcatggtg
     5701 ggcggcaaga acccaaggtc ttgaggcctt cgctaatgcg ggaaagctct tattcgggtg
     5761 agatgggctg gggcaccatc tggggaccct gacgtgaagt ttgtcactga ctggagaact
     5821 cggtttgtcg tctgttgcgg gggcggcagt tatggcggtg ccgttgggca gtgcacccgt
     5881 acctttggga gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat
     5941 gcagggtggg gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag
     6001 ggttcgggcc tagggtaggc tctcctgaat cgacaggcgc cggacctctg gtgaggggag
     6061 ggataagtga ggcgtcagtt tctttggtcg gttttatgta cctatcttct taagtagctg
     6121 aagctccggt tttgaactat gcgctcgggg ttggcgagtg tgttttgtga agttttttag
     6181 gcaccttttg aaatgtaatc atttgggtca atatgtaatt ttcagtgtta gactagtaaa
     6241 ttgtccgcta aattctggcc gtttttggct tttttgttag acgaagcttg ggctgcaggt
     6301 cgactctaga atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     6361 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     6421 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     6481 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     6541 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     6601 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     6661 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     6721 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     6781 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     6841 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     6901 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     6961 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     7021 gtacaaggga tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga
     7081 gaatcctgga ccgatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     7141 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     7201 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     7261 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     7321 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     7381 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     7441 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtgtcgcccg accaccaggg
     7501 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     7561 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     7621 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     7681 gcccggtgcc taaagttgat aatagataac ttcgtatagc atacattata cgaagttatg
     7741 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     7801 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     7861 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     7921 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     7981 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     8041 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     8101 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     8161 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     8221 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     8281 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     8341 ccgcctcccc gcatcgatac cgtcgacctc gagacctaga aaaacatgga gcaatcacaa
     8401 gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg
     8461 aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg
     8521 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     8581 gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc
     8641 agaactacac accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc
     8701 tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt
     8761 tacaccctgt gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt
     8821 ttgacagccg cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt
     8881 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     8941 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     9001 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg
     9061 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     9121 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     9181 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     9241 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     9301 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     9361 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     9421 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     9481 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     9541 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     9601 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     9661 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     9721 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     9781 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     9841 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     9901 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     9961 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
    10021 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
    10081 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
    10141 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata
    10201 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
    10261 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
    10321 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
    10381 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
    10441 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
    10501 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
    10561 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
    10621 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
    10681 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
    10741 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
    10801 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
    10861 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
    10921 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
    10981 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
    11041 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
    11101 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
    11161 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
    11221 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
    11281 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
    11341 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
    11401 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
    11461 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
    11521 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
    11581 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
    11641 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
    11701 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
    11761 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
    11821 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
    11881 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
    11941 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
    12001 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
    12061 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
    12121 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
    12181 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
    12241 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
    12301 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
    12361 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
    12421 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
    12481 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
    12541 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
    12601 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
    12661 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
    12721 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
    12781 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
    12841 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
    12901 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
    12961 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
    13021 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
    13081 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
    13141 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
    13201 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
    13261 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
    13321 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
    13381 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
    13441 ccattgacgt caataatgac gtatgt
//
