LOCUS       RB1-TP53-LRG-EGF        7850 bp DNA     circular SYN 15-AUG-2024
DEFINITION  Lentiviral introduction of sgRNA constitute expression linked with 
            GFP marker into mammalian cell line..
ACCESSION   .
VERSION     .
KEYWORDS    LRG (Lenti_sgRNA_EFS_GFP)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7850)
  AUTHORS   Shi J, Wang E, Milazzo JP, Wang Z, Kinney JB, Vakoc CR
  TITLE     Discovery of cancer drug targets by CRISPR-Cas9 screening of protein
            domains.
  JOURNAL   Nat Biotechnol. 2015 May 11. doi: 10.1038/nbt.3235.
  PUBMED    25961408
REFERENCE   2  (bases 1 to 7850)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 15, 2024 from SnapGene Viewer 6.2.0
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2015 May 11. doi: 10.1038/nbt.3235."
FEATURES             Location/Qualifiers
     source          1..7850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     157..176
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
                     /note="color: black; sequence: GTCTTGTAGTTGCCGTCGTC"
     primer_bind     416..437
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
                     /note="color: black; sequence: CGTCGCCGTCCAGCTCGACCAG"
     CDS             complement(483..506)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /note="color: #cc99b2"
                     /translation="DYKDDDDK"
     regulatory      506..515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     promoter        complement(535..746)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
                     /note="color: #ffffff; direction: LEFT"
     misc_feature    815..932
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
                     /note="color: #ffe4c4"
     misc_RNA        complement(980..1055)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: LEFT"
     misc_feature    1056..1080
                     /label=TP53 guide
                     /note="color: #a6acb3"
     misc_feature    1086..1325
                     /label=U6 promoter
                     /note="color: #ffffff"
     primer_bind     complement(1135..1154)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
                     /note="color: black; sequence: GACTATCATATGCTTACCGT"
     primer_bind     complement(1305..1325)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
                     /note="color: black; sequence: GAGGGCCTATTTCCCATGATT"
     misc_feature    1392..1467
                     /label=gRNA scaffold
                     /note="color: #00ccff"
     misc_feature    1468..1492
                     /label=RB1 guide
                     /note="color: #a6acb3"
     promoter        complement(1497..1737)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(1547..1566)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
                     /note="color: black; sequence: GACTATCATATGCTTACCGT"
     primer_bind     complement(1717..1737)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
                     /note="color: black; sequence: GAGGGCCTATTTCCCATGATT"
     CDS             complement(1915..1959)
                     /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"
                     /note="color: #cc99b2"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2144..2377
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
                     /note="color: #ffe4c4"
     misc_feature    2870..2995
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
                     /note="color: #ffe4c4"
     LTR             3042..3222
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /note="color: #ffe4c4"
     promoter        complement(3223..3449)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(3477..3497)
                     /label=T3
                     /note="T3 promoter, forward primer"
                     /note="color: black; sequence: GCAATTAACCCTCACTAAAGG"
     promoter        complement(3477..3495)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(3516..3532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     primer_bind     complement(3516..3532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
                     /note="color: black; sequence: CAGGAAACAGCTATGAC"
     primer_bind     complement(3529..3551)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
                     /note="color: black; sequence: AGCGGATAACAATTTCACACAGG"
     protein_bind    3540..3556
                     /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)."
                     /note="color: #31849b"
     promoter        complement(3564..3594)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 3564 .. 3570 / #ffffff / -10
                        2: 3571 .. 3588 / #ffffff
                        3: 3589 .. 3594 / #ffffff / -35"
     protein_bind    3609..3630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     primer_bind     complement(3747..3764)
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     rep_origin      complement(3918..4506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     complement(3998..4017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT"
     CDS             complement(4677..5537)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 4677 .. 5468 / #ccffcc
                        2: 5469 .. 5537 / #ccffcc / signal sequence
                     Cleavage site after base 5468"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5300..5319
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: black; sequence: ATAATACCGCGCCACATAGC"
     promoter        complement(5538..5642)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     rep_origin      complement(5668..6123)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     complement(5805..5826)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="color: black; sequence: GTGGACTCTTGTTCCAAACTGG"
     primer_bind     6017..6036
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="color: black; sequence: AGGGAAGAAAGCGAAAGGAG"
     primer_bind     6250..6272
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
                     /note="color: black; sequence: CCCAGTCACGACGTTGTAAAACG"
     primer_bind     6264..6281
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
                     /note="color: black; sequence: TGTAAAACGACGGCCAGT"
     primer_bind     6265..6281
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     primer_bind     6291..6310
                     /label=T7
                     /note="T7 promoter, forward primer"
                     /note="color: black; sequence: TAATACGACTCACTATAGGG"
     promoter        6291..6309
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     rep_origin      6330..6465
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     primer_bind     6369..6380
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
                     /note="color: black; sequence: TATTTATGCAGAGGCCGAGG"
     primer_bind     complement(6384..6403)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
                     /note="color: black; sequence: TATTTATGCAGAGGCCGAGG"
     polyA_signal    6505..6626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(6516..6535)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
                     /note="color: black; sequence: GTGGTTTGTCCAAACTCATC"
     primer_bind     6570..6589
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
                     /note="color: black; sequence: GAAATTTGTGATGCTATTGC"
     LTR             6704..6937
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
                     /note="color: #ffe4c4"
     misc_feature    7009..7597
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /note="color: #ffffff"
     CDS             7115..7126
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2
                     Cleavage site after base 7126"
                     /translation="IEGR"
     primer_bind     7524..7544
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
                     /note="color: black; sequence: CATAGCGTAAAAGGAGCAACA"
     CDS             complement(join(7616..7850,1..482))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                        1: 7616 ..  476 / #05fc14
                        2:  477 ..  479 / #05fc14 / 1a
                        3:  480 ..  482 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7658..7679)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
                     /note="color: black; sequence: CATGGTCCTGCTGGAGTTCGTG"
ORIGIN
        1 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
       61 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
      121 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
      181 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
      241 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg
      301 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
      361 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
      421 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
      481 atcttatcgt catcgtcttt gtaatccatg gtggcggatc ccgcgtcacg acacctgtgt
      541 tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac ttatatacgg
      601 ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt cccagtttac
      661 cccgcgccac cttctctagg caccggttca attgccgacc cctcccccca acttctcggg
      721 gactgtgggc gatgtgcgct ctgcccactg acgggcaccg gagccactcg agccggcgcc
      781 aaagtggatc tctgctgtcc ctgtaataaa cccgaaaatt ttgaattttt gtaatttgtt
      841 tttgtaattc tttagtttgt atgtctgttg ctattatgtc tactattctt tcccctgcac
      901 tgtacccccc aatcccccct tttcttttaa aattgtggat gaatactgcc atttgtctca
      961 agatctagaa ttcaaaaaag caccgactcg gtgccacttt ttcaagttga taacggacta
     1021 gccttatttt aacttgctat ttctagctct aaaacgcatg ggcggcatga accggcggtg
     1081 tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca agttacggta
     1141 agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc aagaaattat
     1201 tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa attaattcta
     1261 attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat gggaaatagg
     1321 ccctctctgg gtcccctcgg ggttgggagg tgggtctgaa acgataatgg tgaatatccc
     1381 tgcccaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt
     1441 ttaacttgct atttctagct ctaaaactcc tgaggaggac ccagagccgg tgtttcgtcc
     1501 tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg
     1561 atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct
     1621 acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc taattatctc
     1681 tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctctct
     1741 gggtcccctc ggggttggga ggtgggtctg aaacgataat ggtgaatatc cctgcctaac
     1801 tctattcact atagaaagta cagcaaaaac tattcttaaa cctaccaagc ctcctactat
     1861 cattatgaat aattttatat accacagcca atttgttatg ttaaaccaat tccacaaact
     1921 tgcccattta tctaattcca ataattcttg ttcattcttt tcttgctggt tttgcgattc
     1981 ttcaattaag gagtgtatta agcttgtgta attgttaatt tctctgtccc actccatcca
     2041 ggtcgtgtga ttccaaatct gttccagaga tttattactc caactagcat tccaaggcac
     2101 agcagtggtg caaatgagtt ttccagagca accccaaatc cccaggagct gttgatcctt
     2161 taggtatctt tccacagcca ggattcttgc ctggagctgc ttgatgcccc agactgtgag
     2221 ttgcaacaga tgctgttgcg cctcaatagc cctcagcaaa ttgttctgct gctgcactat
     2281 accagacaat aattgtctgg cctgtaccgt cagcgtcatt gacgctgcgc ccatagtgct
     2341 tcctgctgct cccaagaacc caaggaacaa agctcctatt cccactgctc ttttttctct
     2401 ctgcaccact cttctctttg ccttggtggg tgctactcct aatggttcaa tttttactac
     2461 tttatattta tataattcac ttctccaatt gtccctcata tctcctcctc caggtctgaa
     2521 gatcagcggc cgcttgctgt gcggtggtct tacttttgtt ttgctcttcc tctatcttgt
     2581 ctaaagcttc cttggtgtct tttatctcta tcctttgatg cacacaatag agggttgcta
     2641 ctgtattata taatgatcta agttcttctg atcctgtctg aagggatggt tgtagctgtc
     2701 ccagtatttg tctacagcct tctgatgttt ctaacaggcc aggattaact gcgaatcgtt
     2761 ctagctccct gcttgcccat actatatgtt ttaatttata ttttttcttt ccccctggcc
     2821 ttaaccgaat tttttcccat cgcgatctaa ttctcccccg cttaatactg acgctctcgc
     2881 acccatctct ctccttctag cctccgctag tcaaaatttt tggcgtactc accagtcgcc
     2941 gcccctcgcc tcttgccgtg cgcgcttcag caagccgagt cctgcgtcga gagagctcct
     3001 ctggtttccc tttcgctttc aagtccctgt tcgggcgcca ctgctagaga ttttccacac
     3061 tgactaaaag ggtctgaggg atctctagtt accagagtca cacaacagac gggcacacac
     3121 tacttgaagc actcaaggca agctttattg aggcttaagc agtgggttcc ctagttagcc
     3181 agagagctcc caggctcaga tctggtctaa ccagagagac ccgtttatgt atcgagctag
     3241 gcacttaaat acaatatctc tgcaatgcgg caattcagtg gttcgtccaa tccatgtcag
     3301 acccgtctgt tgccttccta ataaggcacg atcgtaccac cttacttcca ccaatcggca
     3361 tgcacggtgc tttttctctc cttgtaaggc atgttgctaa ctcatcgtta ccatgttgca
     3421 agactacaag agtattgcat aagactacat taagcttgca gctccagctt ttgttccctt
     3481 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3541 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3601 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3661 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3721 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3781 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3841 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3901 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3961 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4021 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4081 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4141 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4201 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4261 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4321 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4381 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4441 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4501 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4561 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4621 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4681 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4741 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4801 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4861 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4921 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4981 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5041 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5101 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5161 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5221 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5281 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5341 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5401 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5461 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5521 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5581 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5641 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     5701 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     5761 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     5821 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     5881 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     5941 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     6001 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     6061 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     6121 cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     6181 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     6241 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact
     6301 cactataggg cgaattgggt acgtccctag gcctccaaaa aagcctcctc actacttctg
     6361 gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc
     6421 atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg
     6481 cgggatagct agagccagac atgataagat acattgatga gtttggacaa accacaacta
     6541 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     6601 ccattataag ctgcaataaa caagttcctc tcactctctg atattcattt ctttgcaagt
     6661 tataaatact gaataataag atgacatgaa ctactatacg tactgctaga gattttccac
     6721 actgactaaa agggtctgag ggatctctag ttaccagagt cacacaacag acgggcacac
     6781 actacttgaa gcactcaagg caagctttat tgaggcttaa gcagtgggtt ccctagttag
     6841 ccagagagct cccaggctca gatctggtct aaccagagag acccagtaca agcaaaaagc
     6901 agatcttgtc ttcgttggga gtgaattagc ccttccagtc cccccttttc ttttaaaaag
     6961 tggctaagat ctacagctgc cttgtaagtc attggtctta aagtcgacgc ggggaggcgg
     7021 cccaaaggga gatccgactc gtctgagggc gaaggcgaag acgcggaaga ggccgcagag
     7081 ccggcagcag gccgcgggaa ggaaggtccg ctggattgag ggccgaaggg acgtagcaga
     7141 aggacgtccc gcgcagaatc caggtggcaa cacaggcgag cagccaagga aaggacgatg
     7201 atttccccga caacaccacg gaattgtcag tgcccaacag ccgagcccct gtccagcagc
     7261 gggcaaggca ggcggcgatg agttccgccg tggcaatagg gagggggaaa gcgaaagtcc
     7321 cggaaaggag ctgacaggtg gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca
     7381 cagtgcacac cacgccacgt tgcctgacaa cgggccacaa ctcctcataa agagacagca
     7441 accaggattt atacaaggag gagaaaatga aagccatacg ggaagcaata gcatgataca
     7501 aaggcattaa agcagcgtat ccacatagcg taaaaggagc aacatagtta agaataccag
     7561 tcaatctttc acaaattttg taatccagag gttgattgtc gacttaacgc gtctacttgt
     7621 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
     7681 gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt
     7741 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct
     7801 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
//
