LOCUS       sequence-406653-       10248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation, inducible bicistronic lentiviral plasmid for 
            expression of human connexin 26 variant p.G45E and GCaMP6s.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10248)
  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 10248)
  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-Cx26G45E-IRES-GCaMP6s
FEATURES             Location/Qualifiers
     source          1..10248
                     /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(964..1538,1539..1541,1542..1550)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1131..1148)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1358..1377
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1551..1559,1560..1577,1578..1580,1581..1613,1614..1616,
                     1617..1640,1641..1667,1668..1724,1725..1730,1731..2000,
                     2001..2018,2019..2453,2454..2459,2460..2903)
                     /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      1647..1656
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1937..1958
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2064..2085)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2325..2344)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        2910..3420
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3339..3357
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3430..4026
                     /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(3430..3449)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3926..3946
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4027..4080
                     /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(4081..4698,4699..4704,4705..4740,4741..4743,4744..4779,
                     4780..4782,4783..4818,4819..4827)
                     /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(4867..4887)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5285..5296)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5501..5734
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5806..5927
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5843..5862)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5897..5916
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5967..6102
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        6102..6173
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6174..6242,6243..7034)
                     /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(6392..6411)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7205..7793
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7694..7713
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7947..7964
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8081..8102
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8117..8122,8123..8140,8141..8147)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8155..8171
                     /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     8160..8182
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8179..8195
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8179..8195
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8214..8234
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8216..8234
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        8262..8488
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             8489..8669
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    8716..8841
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    9334..9567
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             9752..9796
                     /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    10009..10126
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(10180..10248,1..246)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    10188..10206
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10224..10242
                     /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 gcatggattg gggcacgctg cagacgatcc tggggggtgt
      301 gaacaaacac tccaccagca ttggaaagat ctggctcacc gtcctcttca tttttcgcat
      361 tatgatcctc gttgtggctg caaaggaggt gtgggaagat gagcaggccg actttgtctg
      421 caacaccctg cagccaggct gcaagaacgt gtgctacgat cactacttcc ccatctccca
      481 catccggcta tgggccctgc agctgatctt cgtgtccacg ccagcgctcc tagtggccat
      541 gcacgtggcc taccggagac atgagaagaa gaggaagttc atcaaggggg agataaagag
      601 tgaatttaag gacatcgagg agatcaaaac ccagaaggtc cgcatcgaag gctccctgtg
      661 gtggacctac acaagcagca tcttcttccg ggtcatcttc gaagccgcct tcatgtacgt
      721 cttctatgtc atgtacgacg gcttctccat gcagcggctg gtgaagtgca acgcctggcc
      781 ttgtcccaac actgtggact gctttgtgtc ccggcccacg gagaagactg tcttcacagt
      841 gttcatgatt gcagtgtctg gaatttgcat cctgctgaat gtcactgaat tgtgttattt
      901 gctaattaga tattgttctg ggaagtcaaa aaagccagtt taagaattct taattaaacg
      961 cgtcccctct ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg
     1021 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc
     1081 ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa
     1141 aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag
     1201 acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg
     1261 cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg
     1321 ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa
     1381 caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg
     1441 gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca
     1501 cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc atgggttctc
     1561 atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg ggtcgggatc
     1621 tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt cgtaagtgga
     1681 ataagacagg tcacgcagtc agagctatag gtcggctgag ctcactcgag aacgtctata
     1741 tcaaggccga caagcagaag aacggcatca aggcgaactt ccacatccgc cacaacatcg
     1801 aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc ggcgacggcc
     1861 ccgtgctgct gcccgacaac cactacctga gcgtgcagtc caaactttcg aaagacccca
     1921 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1981 gcatggacga gctgtacaag ggcggtaccg gagggagcat ggtgagcaag ggcgaggagc
     2041 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2101 tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2161 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     2221 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2281 ccatgcccga aggctacatc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2341 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2401 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacctgccgg
     2461 accaactgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta tttgacaagg
     2521 acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct ctggggcaga
     2581 accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac ggtgacggca
     2641 caatcgactt ccctgagttc ctgacaatga tggcaagaaa aatgaaatac agggacacgg
     2701 aagaagaaat tagagaagcg ttcggtgtgt ttgataagga tggcaatggc tacatcagtg
     2761 cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat gaagaggttg
     2821 atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac gaagagtttg
     2881 tacaaatgat gacagcgaag tagggatccg gggttggggt tgcgcctttt ccaaggcagc
     2941 cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc
     3001 gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat cttcgccgct
     3061 acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga aggttccttg
     3121 cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac gtctcactag taccctcgca
     3181 gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt ggccaatagc
     3241 ggctgctcag cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg
     3301 tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct
     3361 ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct ctctccccag
     3421 caattcacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     3481 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     3541 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     3601 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     3661 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     3721 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     3781 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     3841 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     3901 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     3961 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     4021 ggtgccgaag gtagaggttc tctcctcact tgtggtgatg ttgaagaaaa ccctggtcca
     4081 atgtctagac tggacaagag caaagtcata aacggagctc tggaattact caatggtgtc
     4141 ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc
     4201 ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg
     4261 gacaggcatc atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg
     4321 aacaacgcca agtcataccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat
     4381 ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg
     4441 tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt
     4501 acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca
     4561 cctaccaccg attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccggcag
     4621 ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag
     4681 ctaaagtgcg aaagcggcgg gccgaccgac gcccttgacg attttgactt agacatgctc
     4741 ccagccgatg cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat
     4801 tttgaccttg acatgctccc cgggtaacta agtaaggatc gatccaagat atcgtattct
     4861 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     4921 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     4981 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     5041 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     5101 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     5161 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt
     5221 tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     5281 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     5341 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     5401 gcctgtttcg cctcggcgtc cggactagag gtacctttaa gaccaatgac ttacaaggca
     5461 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     5521 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     5581 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     5641 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     5701 agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt
     5761 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca
     5821 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5881 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
     5941 tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     6001 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     6061 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctttcaaat atgtatccgc
     6121 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6181 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6241 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6301 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6361 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6421 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6481 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6541 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6601 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6661 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6721 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6781 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6841 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6901 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6961 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7021 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7081 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7141 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7201 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7261 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7321 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     7381 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7441 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7501 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7561 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7621 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7681 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7741 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7801 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7861 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7921 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     7981 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     8041 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     8101 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     8161 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     8221 accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact
     8281 cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa
     8341 gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca
     8401 acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt
     8461 atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg
     8521 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     8581 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     8641 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
     8701 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
     8761 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
     8821 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
     8881 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
     8941 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     9001 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     9061 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     9121 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     9181 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     9241 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     9301 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     9361 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     9421 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     9481 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     9541 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     9601 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     9661 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     9721 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     9781 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     9841 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     9901 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     9961 gaggggacaa ttctcgacct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa
    10021 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
    10081 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
    10141 ggacagcaga gatccacttt ggccgcgaat cgatatgtcg agtttactcc ctatcagtga
    10201 tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgatg
//
