LOCUS       sequence-371308-       10887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation, inducible bicistronic lentiviral plasmid for 
            expression of human connexin 46 and GCaMP6s.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10887)
  AUTHORS   Nardin C, Tettey-Matey A, Donati V, Marazziti D, Di Pietro C, Peres 
            C, Raspa M, Zonta F, Yang G, Gorelik M, Singh S, Cardarelli L, Sidhu
            SS, Mammano F
  TITLE     A Quantitative Assay for Ca(2+) Uptake through Normal and 
            Pathological Hemichannels.
  JOURNAL   Int J Mol Sci. 2022 Jun 30;23(13). pii: ijms23137337. doi: 
            10.3390/ijms23137337.
  PUBMED    35806342
REFERENCE   2  (bases 1 to 10887)
  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: "Int J Mol 
            Sci."; date: "2022-06-30"; volume: "23(13). pii: ijms23137337. doi";
            pages: " 10.3390/ijms23137337"
COMMENT     Sequence Label: pCW57-Cx46-IRES-GCaMP6s
FEATURES             Location/Qualifiers
     source          1..10887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    11..29
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    47..65
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    83..101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    118..136
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    154..172
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     219..243
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     misc_feature    join(1603..2177,2178..2180,2181..2189)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1770..1787)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1997..2016
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2190..2198,2199..2216,2217..2219,2220..2252,2253..2255,
                     2256..2279,2280..2306,2307..2363,2364..2369,2370..2639,
                     2640..2657,2658..3092,3093..3098,3099..3542)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      2286..2295
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2576..2597
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2703..2724)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2964..2983)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        3549..4059
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3978..3996
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4069..4665
                     /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(4069..4088)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4565..4585
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4666..4719
                     /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(4720..5337,5338..5343,5344..5379,5380..5382,5383..5418,
                     5419..5421,5422..5457,5458..5466)
                     /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(5506..5526)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5924..5935)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6140..6373
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6445..6566
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6482..6501)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6536..6555
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6606..6741
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        6741..6812
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6813..6881,6882..7673)
                     /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(7031..7050)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7844..8432
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8333..8352
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8586..8603
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8720..8741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8756..8761,8762..8779,8780..8786)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8794..8810
                     /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     8799..8821
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8818..8834
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8818..8834
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8853..8873
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8855..8873
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        8901..9127
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             9128..9308
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    9355..9480
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    9973..10206
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             10391..10435
                     /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    10648..10765
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(10819..10887,1..246)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    10827..10845
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10863..10881
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat
       61 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttatcc
      121 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt
      181 cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag
      241 atcgcctgga gaattggcta gcaccggtat gcatggccgg ccgaattcat gggcgactgg
      301 agctttctgg gaagactctt agaaaatgca caggagcact ccacggtcat cggcaaggtt
      361 tggctgaccg tgctgttcat ctttcgcatc ttggtgctgg gggccgcggc ggaggacgtg
      421 tggggcgatg agcagtcaga cttcacctgc aacacccagc agccgggctg cgagaacgtc
      481 tgctacgaca gggccttccc catctcccac atccgcttct gggcgctgca gatcatcttc
      541 gtgtccacgc ccaccctcat ctacctgggc cacgtgctgc acatcgtgcg catggaagag
      601 aagaagaaag agagggagga ggaggagcag ctgaagagag agagccccag ccccaaggag
      661 ccaccgcagg acaatccctc gtcgcgggac gaccgcggca gggtgcgcat ggccggggcg
      721 ctgctgcgga cctacgtctt caacatcatc ttcaagacgc tgttcgaggt gggcttcatc
      781 gccggccagt actttctgta cggcttcgag ctgaagccgc tctaccgctg cgaccgctgg
      841 ccctgcccca acacggtgga ctgcttcatc tccaggccca cggagaagac catcttcatc
      901 atcttcatgc tggcggtggc ctgcgcgtcc ctgctgctca acatgctgga gatctaccac
      961 ctgggctgga agaagctcaa gcagggcgtg accagccgcc tcggcccgga cgcctccgag
     1021 gccccgctgg ggacagccga tcccccgccc ctgcccccca gctcccggcc gcccgccgtt
     1081 gccatcgggt tcccacccta ctatgcgcac accgctgcgc ccctgggaca ggcccgcgcc
     1141 gtgggctacc ccggggcccc gccaccagcc gcggacttca aactgctagc cctgaccgag
     1201 gcgcgcggaa agggccagtc cgccaagctc tacaacggcc accaccacct gctgatgact
     1261 gagcagaact gggccaacca ggcggccgag cggcagcccc cggcgctcaa ggcttacccg
     1321 gcagcgtcca cgcctgcagc ccccagcccc gtcggcagca gctccccgcc actcgcgcac
     1381 gaggctgagg cgggcgcggc gcccctgctg ctggatggga gcggcagcag tctggagggg
     1441 agcgccctgg cagggacccc cgaggaggag gagcaggccg tgaccaccgc ggcccagatg
     1501 caccagccgc ccttgcccct cggagaccca ggtcgggcca gcaaggccag cagggccagc
     1561 agcgggcggg ccagaccgga ggacttggcc atctagacgc gtcccctctc cctccccccc
     1621 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     1681 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     1741 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     1801 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     1861 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     1921 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     1981 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     2041 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt
     2101 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     2161 aaaaacacga tgataatatg gccacaacca tgggttctca tcatcatcat catcatggta
     2221 tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat gacgataagg
     2281 atctcgccac catggtcgac tcatcacgtc gtaagtggaa taagacaggt cacgcagtca
     2341 gagctatagg tcggctgagc tcactcgaga acgtctatat caaggccgac aagcagaaga
     2401 acggcatcaa ggcgaacttc cacatccgcc acaacatcga ggacggcggc gtgcagctcg
     2461 cctaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2521 actacctgag cgtgcagtcc aaactttcga aagaccccaa cgagaagcgc gatcacatgg
     2581 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     2641 gcggtaccgg agggagcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     2701 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggtg
     2761 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2821 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     2881 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacatcc
     2941 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     3001 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     3061 gcaacatcct ggggcacaag ctggagtaca acctgccgga ccaactgact gaagagcaga
     3121 tcgcagaatt taaagaggct ttctccctat ttgacaagga cggggatggg acaataacaa
     3181 ccaaggagct ggggacggtg atgcggtctc tggggcagaa ccccacagaa gcagagctgc
     3241 aggacatgat caatgaagta gatgccgacg gtgacggcac aatcgacttc cctgagttcc
     3301 tgacaatgat ggcaagaaaa atgaaataca gggacacgga agaagaaatt agagaagcgt
     3361 tcggtgtgtt tgataaggat ggcaatggct acatcagtgc agcagagctt cgccacgtga
     3421 tgacaaacct tggagagaag ttaacagatg aagaggttga tgaaatgatc agggaagcag
     3481 acatcgatgg ggatggtcag gtaaactacg aagagtttgt acaaatgatg acagcgaagt
     3541 agggatccgg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc
     3601 ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc
     3661 ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg
     3721 acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg
     3781 tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca
     3841 atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc
     3901 gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc
     3961 ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc
     4021 ggctccctcg ttgaccgaat caccgacctc tctccccagc aattcaccat gaccgagtac
     4081 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     4141 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     4201 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     4261 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     4321 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     4381 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     4441 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     4501 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     4561 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     4621 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgccgaagg tagaggttct
     4681 ctcctcactt gtggtgatgt tgaagaaaac cctggtccaa tgtctagact ggacaagagc
     4741 aaagtcataa acggagctct ggaattactc aatggtgtcg gtatcgaagg cctgacgaca
     4801 aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca cgtgaagaac
     4861 aagcgggccc tgctcgatgc cctgccaatc gagatgctgg acaggcatca tacccacttc
     4921 tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa gtcataccgc
     4981 tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg cccaacagag
     5041 aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg cttctccctg
     5101 gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg cgtattggag
     5161 gaacaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga ttctatgccc
     5221 ccacttctga gacaagcaat tgagctgttc gaccggcagg gagccgaacc tgccttcctt
     5281 ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga aagcggcggg
     5341 ccgaccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc ccttgacgac
     5401 tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga catgctcccc
     5461 gggtaactaa gtaaggatcg atccaagata tcgtattctt aactatgttg ctccttttac
     5521 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     5581 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     5641 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     5701 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     5761 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     5821 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     5881 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     5941 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     6001 ccctcagacg agtcggatct ccctttgggc cgcctccccg cctgtttcgc ctcggcgtcc
     6061 ggactagagg tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     6121 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     6181 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     6241 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     6301 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     6361 aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     6421 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     6481 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     6541 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     6601 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     6661 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     6721 gaggaggctt ttttggaggc ctttcaaata tgtatccgct catgagacaa taaccctgat
     6781 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6841 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6901 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6961 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     7021 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     7081 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     7141 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     7201 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     7261 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     7321 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     7381 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     7441 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     7501 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     7561 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     7621 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     7681 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     7741 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     7801 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     7861 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     7921 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     7981 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     8041 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     8101 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     8161 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     8221 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     8281 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     8341 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     8401 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     8461 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     8521 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     8581 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     8641 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     8701 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     8761 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     8821 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
     8881 agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt
     8941 aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt
     9001 ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat
     9061 tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata
     9121 cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     9181 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     9241 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     9301 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
     9361 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
     9421 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
     9481 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
     9541 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
     9601 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     9661 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     9721 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     9781 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
     9841 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     9901 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     9961 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
    10021 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
    10081 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
    10141 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
    10201 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
    10261 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
    10321 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
    10381 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
    10441 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
    10501 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
    10561 atattcacca ttatcgtttc agacccacct cccaaccccg aggggacaat tctcgacctc
    10621 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
    10681 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
    10741 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
    10801 gccgcgaatc gatatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
    10861 actccctatc agtgatagag aacgatg
//
