LOCUS       sequence-204854-        7298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Functional studies using RNAi; rescue experiments; substituting 
            protein for its different form. miRNA cassette with nuclear mCherry 
            expression marker; your protein of interest with N-terminal EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7298)
  AUTHORS   Szczesny RJ, Kowalska K, Klosowska-Kosicka K, Chlebowski A, Owczarek
            EP, Warkocki Z, Kulinski TM, Adamska D, Affek K, Jedroszkowiak A, 
            Kotrys AV, Tomecki R, Krawczyk PS, Borowski LS, Dziembowski A
  TITLE     Versatile approach for functional analysis of human proteins and 
            efficient stable cell line generation using FLP-mediated 
            recombination system.
  JOURNAL   PLoS One. 2018 Mar 28;13(3):e0194887. doi: 
            10.1371/journal.pone.0194887. eCollection 2018.
  PUBMED    29590189
REFERENCE   2  (bases 1 to 7298)
  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: "PLoS One.";
            date: "2018-03-28"; volume: "13(3):e0194887. doi"; pages: " 
            10.1371/journal.pone.0194887. eCollection 2018"
COMMENT     Sequence Label: pKK-RNAi-nucCHERRYmiR-EGFP-TEV
FEATURES             Location/Qualifiers
     source          1..7298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(6..26)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    421..469
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     494..513
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     ncRNA           complement(585..626)
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(690..716)
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(723..764)
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(828..854)
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(861..902)
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(966..992)
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     CDS             complement(1038..1058)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1062..1082)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1086..1106)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1119..1826)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1394..1413)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1663..1681
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    1840..1858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1861..1879
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(1881..2084)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        2264..2315
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    2317..2335
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2338..2356
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(2380..2382,2383..2385,2386..3096)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2380..2382,2383..2385,2386..3096)
                     /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(2425..2446)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2686..2705)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3033..3054
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3103..3123
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3164..3184
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3233..3250)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3239..3463
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3596..3615)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3747..3794
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3802..4824
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3802..4824
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4954..5075
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4991..5010)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5045..5064
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5124..5140)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5124..5140)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5137..5159)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5148..5164
                     /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(5172..5178,5179..5196,5197..5202))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5217..5238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5355..5372)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5526..6114)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5606..5625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6285..7076,7077..7145))
                     /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     6908..6927
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7146..7250)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
       61 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
      121 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
      181 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc tccatcgttc
      241 agatccttat cgatgctatg gcagggcctg ccgccccgac gttggctgcg agccctgggc
      301 cttcacccga acttgggggg tggggtgggg aaaaggaaga aacgcgggcg tattggcccc
      361 aatggggtca cggtggggta tcgacagagt gccagccctg ggaccgaacc ccgcgtttat
      421 gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg
      481 gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatca gcgaaccgcg
      541 ggccctctag atcactagat atgacgcgtg cggccgcaga tctgggccat ttgttccatg
      601 tgagtgctag taacaggcct tgtgtcctga aatgtactgc gtggagacgt cagtcagtgg
      661 ccaaaacgtc tccacgcgca gtacatttca gcatacagcc ttcagcaagc ctccaggatc
      721 tgggccattt gttccatgtg agtgctagta acaggccttg tgtcctgaaa tgtactgcgt
      781 ggagacgtca gtcagtggcc aaaacgtctc cacgcgcagt acatttcagc atacagcctt
      841 cagcaagcct ccaggatctg ggccatttgt tccatgtgag tgctagtaac aggccttgtg
      901 tcctgaaatg tactgcgtgg agacgtcagt cagtggccaa aacgtctcca cgcgcagtac
      961 atttcagcat acagccttca gcaagcctcc aggatcactt aagactagcg ctccaattgc
     1021 actacctcct cttactacac cttgcgtttc ttctttgggt cgacctttct cttcttttta
     1081 gggtccacct tccgcttttt cttggggtct gctgcggcct tgtacagctc gtccatgccg
     1141 ccggtggagt ggcggccctc ggcgcgttcg tactgttcca cgatggtgta gtcctcgttg
     1201 tgggaggtga tgtccaactt gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc
     1261 ttggccttgt aggtggtctt gacctcagcg tcgtagtggc cgccgtcctt cagcttcagc
     1321 ctctgcttga tctcgccctt cagggcgccg tcctcggggt acatccgctc ggaggaggcc
     1381 tcccagccca tggtcttctt ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc
     1441 agcttcacct tgtagatgaa ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc
     1501 acgccgccgt cctcgaagtt catcacgcgc tcccacttga agccctcggg gaaggacagc
     1561 ttcaagtagt cggggatgtc ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac
     1621 tgaggggaca ggatgtccca ggcgaagggc agggggccac ccttggtcac cttcagcttg
     1681 gcggtctggg tgccctcgta ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg
     1741 ccgttcacgg agccctccat gtgcaccttg aagcgcatga actccttgat gatggccatg
     1801 ttatcctcct cgcccttgct caccatggtc ctaggacgat ctctatcact gatagggaga
     1861 tctctatcac tgatagggag agctctgctt atatagacct cccaccgtac acgcctaccg
     1921 cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg
     1981 tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca
     2041 aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg
     2101 actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa
     2161 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
     2221 acttgatgta ctctagaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     2281 ggcgtgtacg gtgggaggtc tatataagca gagctctccc tatcagtgat agagatctcc
     2341 ctatcagtga tagagatcgt cggatctaag cttggtacca tggtgagcaa gggcgaggag
     2401 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2461 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     2521 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2581 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2641 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2701 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2761 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2821 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2881 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2941 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     3001 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     3061 gccgggatca ctctcggcat ggacgagctg tacaagggat ccgaaaacct gtacttccaa
     3121 ggaaccggtg gtcacctgta caatcgatcc tgcaggagct agcgagaatt tgtattttca
     3181 gggtgatatc cagcacagtg gcagctgctc gaggtttaaa cccgctgatc agcctcgact
     3241 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     3301 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgtatc gcattgtctg
     3361 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     3421 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     3481 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     3541 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3601 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3661 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3721 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     3781 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     3841 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     3901 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     3961 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     4021 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     4081 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     4141 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     4201 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     4261 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     4321 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     4381 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     4441 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     4501 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     4561 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     4621 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     4681 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     4741 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     4801 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     4861 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4921 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4981 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5041 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5101 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5161 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5221 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5281 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5341 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5401 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5461 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5521 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5581 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5641 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5701 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5761 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5821 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5881 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5941 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6001 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6061 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6121 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6181 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6241 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6301 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6361 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6421 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6481 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6541 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6601 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6661 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6721 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6781 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6841 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6901 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6961 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7021 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7081 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7141 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7201 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7261 cgaaaagtgc cacctgacgt cgacggatcg ggagatcc
//
