LOCUS       sequence-310820-       11917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses customizable S. Pyogenes sgRNA from U6 promoter and 
            PuroR-P2A-EGFP from EF-1a promoter. Stuffer contains mRFP from a 
            bacterial promoter, enabling simple visual quality control step of 
            sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11917)
  AUTHORS   Panda SK, Wigerblad G, Jiang L, Jimenez-Andrade Y, Iyer VS, Shen Y, 
            Boddul SV, Guerreiro-Cacais AO, Raposo B, Kasza Z, Wermeling F
  TITLE     IL-4 controls activated neutrophil FcgammaR2b expression and 
            migration into inflamed joints.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Jan 24. pii: 1914186117. doi: 
            10.1073/pnas.1914186117.
  PUBMED    31980518
REFERENCE   2  (bases 1 to 11917)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2020 Jan 24. pii: 1914186117. doi: 
            10.1073/pnas.1914186117."
COMMENT     Sequence Label: LentiGuide-Puro-P2A-EGFP_mRFPstuf
FEATURES             Location/Qualifiers
     source          1..11917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(683..688,689..706,707..713)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    721..737
                     /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)."
     RBS             755..766
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             773..1450
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1495..1566
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_RNA        2844..2919
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2975..3092
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3231..4409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3462..4400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4357..4377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4422..5018
                     /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(4422..4441)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4918..4938
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5028..5084
                     /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             join(5085..5087,5088..5801)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5085..5087,5088..5798)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5127..5148)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5388..5407)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5735..5756
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5817..6405
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5870..5890)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6288..6299)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6477..6710
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6788..6909
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6825..6844)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6879..6898
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6949..7084
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7104..7123)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7105..7123)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7133..7150)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7133..7149)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7142..7164)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7291..7746
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7378..7397)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7588..7609
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7772..7876
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7877..7945,7946..8737)
                     /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     complement(8095..8114)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8908..9496
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9397..9416
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9650..9667
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9784..9805
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9820..9825,9826..9843,9844..9850)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9858..9874
                     /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)."
     primer_bind     9882..9898
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9882..9898
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9917..9937
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9919..9937
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        9965..10191
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             10192..10372
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    10419..10544
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11037..11270
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             11455..11499
                     /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"
     promoter        join(11677..11917,1..8)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     11677..11697
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     11848..11867
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gaaacaccgg agacggttgt aaatgagcac acaaaataca catgctaaaa tattatattc
       61 tatgaccttt ataaaatcaa ccaaaatctt ctttttaata actttagtat caataattag
      121 aatttttatg ttcctttttg caaactttta ataaaaatga gcaaaataaa aaaacgctag
      181 ttttagtaac tcgcgttgtt ttcttcacct ttaataatag ctactccacc acttgttcct
      241 aagcggtcag ctcctgcttc aatcattttt tgagcatctt caaatgttct aactccacca
      301 gctgctttaa ctaaagcatt gtctttaaca actgacttca ttagtttaac atcttcaaat
      361 gttgcacctg attttgaaaa tcctgttgat gttttaacaa attctaatcc agcttcaaca
      421 gctatttcac aagctttcat gatttcttct tttgttaata aacaattttc cataatacat
      481 ttaacaacat gtgatccagc tgcttttttt acagctttca tgtcttctaa aactaattca
      541 taatttttgt cttttaatgc accaatattt aataccatat caatttctgt tgcaccatct
      601 ttaattgctt cagaaactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      661 tcacacatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      721 ttgtgagcgg ataacaattt cacacatact agagaaagag gagaaatact agatggcttc
      781 ctccgaggac gttatcaaag agttcatgcg tttcaaagtt cgtatggaag gttccgttaa
      841 cggtcacgag ttcgaaatcg aaggtgaagg tgaaggtcgt ccgtacgaag gtacccagac
      901 cgctaaactg aaagttacca aaggtggtcc gctgccgttc gcttgggaca tcctgtcccc
      961 gcagttccag tacggttcca aagcttacgt taaacacccg gctgacatcc cggactacct
     1021 gaaactgtcc ttcccggaag gtttcaaatg ggaacgtgtt atgaacttcg aggacggtgg
     1081 tgttgttacc gttacccagg actcctccct gcaagacggt gagttcatct acaaagttaa
     1141 actgcgtggt accaacttcc cgtccgatgg tccggttatg cagaaaaaaa ccatgggttg
     1201 ggaagcttcc accgaacgta tgtacccgga ggacggtgct ctgaaaggtg aaatcaaaat
     1261 gcgtctgaaa ctgaaagacg gtggtcacta cgacgctgaa gttaaaacca cctacatggc
     1321 taaaaaaccg gttcagctgc cgggtgctta caaaaccgac atcaaactgg acatcacctc
     1381 ccacaacgag gactacacca tcgttgaaca gtacgaacgt gctgaaggtc gtcactccac
     1441 cggtgcttaa taacgctgat agtgctagtg tagatcgcta ctagagccag gcatcaaata
     1501 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     1561 gctctctact agagtcacac tggtgtagct gttgtgcatg cacctagagg aaaacctaca
     1621 acatttgtta ttcctacatt tgtgcctttt aataattctt tacaatagct tgttcaatat
     1681 gaattaacac aaactgttgc aaaatcaaat tcaattgctt catcacataa ttgtttaatt
     1741 tcagctttcg tagcatcttg ttttaataat gtgtgatcta tatatttgtt tagtttcatt
     1801 ttttctccta tatattcatt tttaatttta attctttaat aatttcgtct actttaactt
     1861 tagcgttttg aacagattca ccaacaccta taaaataaat ttttagttta ggttcagttc
     1921 cacttgggcg aacagcaaat catgacttat cttctaaata aaattttagt aagtcttgtc
     1981 ctggcatatt atacattcca tcgatgtagt cttcaacatt aacaacttta agtccagcaa
     2041 tttgagttaa gggtgttgct ctcaatgatt tcattaatgg ttcaattttt aatttctttt
     2101 cttctggttt aaaattcaag tttaaagtga aagtgtaata tgcacccatt tctttaaata
     2161 aatcttctaa atagtctact aatgttttat tttgtttttt ataaaatcaa gcagcctctg
     2221 ctattaatat agaagcttgt attccatctt tatctctagc tgagtcatca attacatatc
     2281 cataactttc ttcataagca aaaacaaaat ttaatccgtt atcttcttct ttagcaattt
     2341 ctctacccat tcatttaaat ccagttaaag tttttacaat attaactcca tatttttcat
     2401 gagcgattct atcacccaaa tcacttgtta caaaacttga atatagagcc ggattttttg
     2461 gaatgctatt taagcgtttt agatttgata attttcaatc aattaaaatt ggtcctgttt
     2521 gatttccatc taatcttaca aaatgaccat catgttttat tgccattcca aatctgtcag
     2581 catctgggtc attcataata ataatatctg catcatgttt aataccatat tcaagcggta
     2641 tttttcatgc aggatcaaat tctggatttg gatttacaac atttttaaat gtttcatctt
     2701 caaatgcatg ctcttcaacc tcaataacgt tatatcctga ttcacgtaat atttttgggg
     2761 taaatttagt tcctgttcca ttaactgcgc taaaaataat ttttaaatct tttttagctt
     2821 cttgctcttt tttgtacgtc tctgttttag agctagaaat agcaagttaa aataaggcta
     2881 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttagaat tcgcgtaact
     2941 agatcttgag acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg
     3001 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat
     3061 tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc
     3121 cactttggcg ccggctcgag ggggcccggg tgcaaagatg gataaagttt taaacagaga
     3181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
     3241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     3301 gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     3361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     3421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
     3481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
     3541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
     3601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
     3661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
     3721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
     3781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
     3841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
     3901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
     3961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     4021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     4081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     4141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     4201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     4261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     4321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     4381 gtttttttct tccatttcag gtgtcgtgac gtacggccac catgaccgag tacaagccca
     4441 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     4501 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     4561 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     4621 cggacgacgg cgccgccgtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     4681 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     4741 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     4801 ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     4861 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     4921 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     4981 gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg atccggcgca acaaacttct
     5041 ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accggtgagc aagggcgagg
     5101 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     5161 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     5221 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     5281 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     5341 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     5401 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     5461 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     5521 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     5581 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     5641 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     5701 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     5761 ccgccgggat cactctcggc atggacgagc tgtacaagta aacgcgttaa gtcgacaatc
     5821 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     5881 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     5941 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     6001 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     6061 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     6121 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     6181 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     6241 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     6301 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     6361 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc
     6421 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     6481 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     6541 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     6601 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     6661 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag
     6721 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
     6781 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     6841 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     6901 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta
     6961 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     7021 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     7081 ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     7141 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     7201 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     7261 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     7321 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     7381 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     7441 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     7501 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     7561 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     7621 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     7681 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     7741 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     7801 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     7861 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     7921 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     7981 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     8041 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     8101 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     8161 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     8221 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     8281 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     8341 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     8401 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     8461 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     8521 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     8581 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     8641 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     8701 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     8761 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     8821 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     8881 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     8941 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     9001 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     9061 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     9121 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     9181 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     9241 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     9301 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     9361 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     9421 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     9481 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     9541 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     9601 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     9661 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     9721 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     9781 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     9841 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     9901 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
     9961 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
    10021 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
    10081 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
    10141 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
    10201 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
    10261 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
    10321 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
    10381 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
    10441 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
    10501 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
    10561 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
    10621 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
    10681 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
    10741 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
    10801 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
    10861 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
    10921 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
    10981 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
    11041 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
    11101 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
    11161 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
    11221 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
    11281 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
    11341 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
    11401 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
    11461 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
    11521 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
    11581 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
    11641 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
    11701 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
    11761 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
    11821 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
    11881 ttcgatttct tggctttata tatcttgtgg aaaggac
//
