LOCUS       sequence-450837-       10116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway cloning of genes of interest with an N-terminal V5-mECFP 
            fusion; generation of stable cells with doxycycline inducible 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10116)
  TITLE     Zaja lab plasmids
REFERENCE   2  (bases 1 to 10116)
  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"
COMMENT     Sequence Label: plix403-V5-N-mECFP
FEATURES             Location/Qualifiers
     source          1..10116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    6..130
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(155..160,161..178,179..185)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             239..898
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(305..324)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1240..1545
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1424..1443
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1586..1710)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1715..1756
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1770..1787
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1797..2307
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2226..2244
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2317..2913
                     /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(2317..2336)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2813..2833
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2914..2967
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(2968..3585,3586..3591,3592..3627,3628..3630,3631..3666,
                     3667..3669,3670..3705,3706..3714)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(3754..3774)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4172..4183)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4388..4621
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4693..4814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4730..4749)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4784..4803
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4854..4989
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4989..5060
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5061..5129,5130..5921)
                     /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(5279..5298)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6092..6680
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6581..6600
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6834..6851
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6968..6989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7004..7009,7010..7027,7028..7034)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7042..7058
                     /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     7047..7069
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7066..7082
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7066..7082
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7101..7121
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7103..7121
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7149..7375
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7376..7556
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7603..7728
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8221..8454
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8639..8683
                     /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    8896..9013
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        9067..9381
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    9075..9093
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9111..9129
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9146..9164
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9182..9200
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9218..9236
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9253..9271
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9289..9307
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     9354..9378
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      9393..9402
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(9399..9401,9402..9404,9405..10115)
                     /codon_start=1
                     /product="enhanced CFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(9444..9465)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(9705..9724)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     10052..10073
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 catcaacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata aatatcaata
       61 tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca caacatatcc
      121 agtcactatg gcggccgcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      181 taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg aagctaaaat
      241 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
      301 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
      361 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
      421 cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga aagacggtga
      481 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
      541 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
      601 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
      661 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
      721 caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga
      781 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt
      841 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaaag
      901 atctggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg ctgatttttg
      961 cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag gtatgctatg
     1021 aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa ggcatatatg
     1081 atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg
     1141 ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg tttattgaaa
     1201 tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc
     1261 tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat tattgacacg
     1321 cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc
     1381 cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat
     1441 atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa
     1501 aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc aggctccctt
     1561 atacacagcc agtctgcagg tcgaccatag tgactggata tgttgtgttt tacagtatta
     1621 tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat cattttacgt
     1681 ttctcgttca gctttcttgt acaaagtggt tgcaggaaag ccaatcccaa acccactcct
     1741 aggtctcgac agtacagcat agaccggtcc accaccacca ccaccactaa ggatccgggg
     1801 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
     1861 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
     1921 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
     1981 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
     2041 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
     2101 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
     2161 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
     2221 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
     2281 gaccgaatca ccgacctctc tccccagcaa ttcaccatga ccgagtacaa gcccacggtg
     2341 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     2401 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     2461 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     2521 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     2581 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     2641 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     2701 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     2761 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     2821 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     2881 cgcacctggt gcatgacccg caagcccggt gccgaaggta gaggttctct cctcacttgt
     2941 ggtgatgttg aagaaaaccc tggtccaatg tctagactgg acaagagcaa agtcataaac
     3001 ggagctctgg aattactcaa tggtgtcggt atcgaaggcc tgacgacaag gaaactcgct
     3061 caaaagctgg gagttgagca gcctaccctg tactggcacg tgaagaacaa gcgggccctg
     3121 ctcgatgccc tgccaatcga gatgctggac aggcatcata cccacttctg ccccctggaa
     3181 ggcgagtcat ggcaagactt tctgcggaac aacgccaagt cataccgctg tgctctcctc
     3241 tcacatcgcg acggggctaa agtgcatctc ggcacccgcc caacagagaa acagtacgaa
     3301 accctggaaa atcagctcgc gttcctgtgt cagcaaggct tctccctgga gaacgcactg
     3361 tacgctctgt ccgccgtggg ccactttaca ctgggctgcg tattggagga acaggagcat
     3421 caagtagcaa aagaggaaag agagacacct accaccgatt ctatgccccc acttctgaga
     3481 caagcaattg agctgttcga ccggcaggga gccgaacctg ccttcctttt cggcctggaa
     3541 ctaatcatat gtggcctgga gaaacagcta aagtgcgaaa gcggcgggcc gaccgacgcc
     3601 cttgacgatt ttgacttaga catgctccca gccgatgccc ttgacgactt tgaccttgat
     3661 atgctgcctg ctgacgctct tgacgatttt gaccttgaca tgctccccgg gtaactaagt
     3721 aaggatcgat ccaagatatc gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3781 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3841 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     3901 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3961 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     4021 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     4081 ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat
     4141 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     4201 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     4261 tcggatctcc ctttgggccg cctccccgcc tgtttcgcct cggcgtccgg actagaggta
     4321 cctttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     4381 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     4441 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     4501 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     4561 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     4621 agtagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca
     4681 gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4741 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4801 atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc
     4861 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4921 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4981 ttggaggcct ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5041 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5101 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5161 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5221 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5281 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5341 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5401 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5461 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5521 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5581 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5641 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5701 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5761 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5821 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5881 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5941 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6001 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6061 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6121 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6181 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6241 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6301 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6361 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6421 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6481 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6541 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6601 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6661 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6721 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6781 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6841 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6901 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6961 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     7021 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     7081 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
     7141 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
     7201 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
     7261 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
     7321 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
     7381 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     7441 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     7501 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     7561 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     7621 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     7681 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     7741 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     7801 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     7861 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     7921 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     7981 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     8041 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     8101 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     8161 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     8221 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     8281 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     8341 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     8401 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     8461 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     8521 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     8581 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     8641 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     8701 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     8761 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     8821 atcgtttcag acccacctcc caaccccgag gggacaattc tcgacctcga gacaaatggc
     8881 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
     8941 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
     9001 aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc cgcgaatcga
     9061 tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag
     9121 tgatagagaa cgatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
     9181 ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt
     9241 atgtcgagtt tatccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg
     9301 atagagaacg tatgtcgagg taggcgtgta cggtgggagg cctatataag cagagctcgt
     9361 ttagtgaacc gtcagatcgc ctggagaatt gggccaccat ggtgagcaag ggcgaggagc
     9421 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     9481 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     9541 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctggg
     9601 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     9661 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     9721 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     9781 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacatca
     9841 gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     9901 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     9961 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc
    10021 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
    10081 ccgggatcac tctcggcatg gacgagctgt acaagt
//
