LOCUS       sequence-462688-       12977 bp DNA     circular SYN 26-FEB-2025
DEFINITION  gRNA cloning vector encoding BFP and puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12977)
  AUTHORS   Liu S, Hamilton MC, Cowart T, Barrera A, Bounds LR, Nelson AC, 
            Dornbaum SF, Riley JW, Doty RW, Allen AS, Crawford GE, Majoros WH, 
            Gersbach CA
  TITLE     Characterization and bioinformatic filtering of ambient gRNAs in 
            single-cell CRISPR screens using CLEANSER.
  JOURNAL   Cell Genom. 2025 Feb 4:100766. doi: 10.1016/j.xgen.2025.100766.
  PUBMED    39914388
REFERENCE   2  (bases 1 to 12977)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 26, 2025 from SnapGene Server 7.0.3
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Genom.
            2025 Feb 4:100766. doi: 10.1016/j.xgen.2025.100766."
FEATURES             Location/Qualifiers
     source          1..12977
                     /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        4600..5811
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5648..5667
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5840..6538
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             6548..6604
                     /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             6605..7204
                     /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(6605..6624)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7101..7121
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(7217..7250)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    7275..7863
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7328..7348)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7746..7757)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7866..7882)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7867..7883)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8388..8568
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(8594..8611)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8600..8824
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8870..9298
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8957..8976)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9167..9188
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9307..9327)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9312..9641
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9492..9627
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9554..9573
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9689..9736
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9755..10129
                     /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    10259..10380
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10296..10315)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10350..10369
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10429..10445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10442..10464)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10453..10469
                     /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(10477..10507)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10522..10543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10660..10677)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10831..11419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10911..10930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(11590..12450)
                     /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     12213..12232
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12451..12555)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12630..12649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(12821..12977,1..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 cgagcgctgc cacgtcagac gaagggcgca
     4681 gcgagcgtcc tgatccttcc gcccggacgc tcaggacagc ggcccgctgc tcataagact
     4741 cggccttaga accccagtat cagcagaagg acattttagg acgggacttg ggtgactcta
     4801 gggcactggt tttctttcca gagagcggaa caggcgagga aaagtagtcc cttctcggcg
     4861 attctgcgga gggatctccg tggggcggtg aacgccgatg attatataag gacgcgccgg
     4921 gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg tttgtggatc
     4981 gctgtgatcg tcacttggtg agtagcgggc tgctgggctg gccggggctt tcgtggccgc
     5041 cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc
     5101 gcgagcaagg ttgccctgaa ctgggggttg gggggagcgc agcaaaatgg cggctgttcc
     5161 cgagtcttga atggaagacg cttgtgaggc gggctgtgag gtcgttgaaa caaggtgggg
     5221 ggcatggtgg gcggcaagaa cccaaggtct tgaggccttc gctaatgcgg gaaagctctt
     5281 attcgggtga gatgggctgg ggcaccatct ggggaccctg acgtgaagtt tgtcactgac
     5341 tggagaactc ggtttgtcgt ctgttgcggg ggcggcagtt atggcggtgc cgttgggcag
     5401 tgcacccgta cctttgggag cgcgcgccct cgtcgtgtcg tgacgtcacc cgttctgttg
     5461 gcttataatg cagggtgggg ccacctgccg gtaggtgtgc ggtaggcttt tctccgtcgc
     5521 aggacgcagg gttcgggcct agggtaggct ctcctgaatc gacaggcgcc ggacctctgg
     5581 tgaggggagg gataagtgag gcgtcagttt ctttggtcgg ttttatgtac ctatcttctt
     5641 aagtagctga agctccggtt ttgaactatg cgctcggggt tggcgagtgt gttttgtgaa
     5701 gttttttagg caccttttga aatgtaatca tttgggtcaa tatgtaattt tcagtgttag
     5761 actagtaaat tgtccgctaa attctggccg tttttggctt ttttgttaga cgaagcttgg
     5821 gctgcaggtc gactctagaa tgagcgagct gattaaggag aacatgcaca tgaagctgta
     5881 catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc
     5941 ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc
     6001 cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca
     6061 gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac
     6121 cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg
     6181 cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca
     6241 gaagaaaaca ctcggctggg aggccttcac cgagaccctg taccccgctg acggcggcct
     6301 ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat
     6361 caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta
     6421 tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca
     6481 cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatgg
     6541 atccggcgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag agaatcctgg
     6601 accgatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     6661 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     6721 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     6781 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     6841 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     6901 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     6961 ggagcccgcg tggttcctgg ccaccgtcgg cgtgtcgccc gaccaccagg gcaagggtct
     7021 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     7081 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     7141 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     7201 ctaaagttga taatagataa cttcgtatag catacattat acgaagttat gaattcgata
     7261 tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     7321 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     7381 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     7441 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     7501 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     7561 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     7621 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     7681 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     7741 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     7801 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     7861 cgcatcgata ccgtcgacct cgagacctag aaaaacatgg agcaatcaca agtagcaata
     7921 cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt
     7981 ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct gtagatctta
     8041 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     8101 atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca
     8161 caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag
     8221 ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg
     8281 tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc
     8341 gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt
     8401 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     8461 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     8521 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa
     8581 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     8641 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     8701 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     8761 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     8821 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     8881 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     8941 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     9001 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     9061 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     9121 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     9181 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     9241 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     9301 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     9361 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     9421 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     9481 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     9541 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     9601 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     9661 tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg
     9721 catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc
     9781 ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt
     9841 ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt
     9901 catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg
     9961 cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc
    10021 ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg
    10081 cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctacg
    10141 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
    10201 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
    10261 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
    10321 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
    10381 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
    10441 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
    10501 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
    10561 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
    10621 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
    10681 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
    10741 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
    10801 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
    10861 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
    10921 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
    10981 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
    11041 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
    11101 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
    11161 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
    11221 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
    11281 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
    11341 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
    11401 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
    11461 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
    11521 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
    11581 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
    11641 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
    11701 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
    11761 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
    11821 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
    11881 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
    11941 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
    12001 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
    12061 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
    12121 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
    12181 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
    12241 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
    12301 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
    12361 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
    12421 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
    12481 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
    12541 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
    12601 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
    12661 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
    12721 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
    12781 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
    12841 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
    12901 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
    12961 tcaataatga cgtatgt
//
