LOCUS       sequence-430341-       10431 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Inducible bicistronic lentiviral plasmid for the simultaneous 
            expression of the D178Y mutant form of Connexin 32 and cytosolic 
            GCaMP6s.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10431)
  AUTHORS   Tettey-Matey A, Donati V, Cimmino C, Di Pietro C, Buratto D, 
            Panarelli M, Reale A, Calistri A, Fornaini MV, Zhou R, Yang G, Zonta
            F, Marazziti D, Mammano F
  TITLE     A fully human IgG1 antibody targeting connexin 32 extracellular 
            domain blocks CMTX1 hemichannel dysfunction in an in vitro model.
  JOURNAL   Cell Commun Signal. 2024 Dec 5;22(1):589. doi: 
            10.1186/s12964-024-01969-0.
  PUBMED    39639332
REFERENCE   2  (bases 1 to 10431)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Commun
            Signal."; date: "2024-12-5"; volume: "22(1):589. doi"; pages: " 
            10.1186/s12964-024-01969-0"
COMMENT     Sequence Label: pCW57-Cx32D178Y-IRES-GCaMP6s
FEATURES             Location/Qualifiers
     source          1..10431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /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    1521..1638
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1692..2006
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1700..1718
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1736..1754
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1771..1789
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1807..1825
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1843..1861
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1878..1896
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1914..1932
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1979..2003
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     misc_feature    join(2907..3481,3482..3484,3485..3493)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3074..3091)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3301..3320
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3494..3502,3503..3520,3521..3523,3524..3556,3557..3559,
                     3560..3583,3584..3610,3611..3667,3668..3673,3674..3943,
                     3944..3961,3962..4396,4397..4402,4403..4846)
                     /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      3590..3599
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3880..3901
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4007..4028)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4268..4287)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        4853..5363
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     5282..5300
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             5373..5969
                     /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(5373..5392)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5869..5889
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5970..6023
                     /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(6024..6641,6642..6647,6648..6683,6684..6686,6687..6722,
                     6723..6725,6726..6761,6762..6770)
                     /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(6810..6830)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7228..7239)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7444..7677
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7749..7870
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7786..7805)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7840..7859
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7910..8045
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        8045..8116
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8117..8185,8186..8977)
                     /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(8335..8354)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9148..9736
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9637..9656
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9890..9907
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10024..10045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10060..10065,10066..10083,10084..10090)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10098..10114
                     /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     10103..10125
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10122..10138
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10122..10138
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10157..10177
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10159..10177
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        10205..10431
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac aattctcgac ctcgagacaa
     1501 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     1561 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     1621 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggccgcga
     1681 atcgatatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
     1741 atcagtgata gagaacgatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga
     1801 gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
     1861 aacgtatgtc gagtttatcc ctatcagtga tagagaacgt atgtcgagtt tactccctat
     1921 cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat ataagcagag
     1981 ctcgtttagt gaaccgtcag atcgcctgga gaattggcta gcaccggtat gcatggccgg
     2041 ccgaattcat gaactggaca ggtttgtaca ccttgctcag tggcgtgaac cggcattcta
     2101 ctgccattgg ccgagtatgg ctctcggtca tcttcatctt cagaatcatg gtgctggtgg
     2161 tggctgcaga gagtgtgtgg ggtgatgaga aatcttcctt catctgcaac acactccagc
     2221 ctggctgcaa cagcgtttgc tatgaccaat tcttccccat ctcccatgtg cggctgtggt
     2281 ccctgcagct catcctagtt tccaccccag ctctcctcgt ggccatgcac gtggctcacc
     2341 agcaacacat agagaagaaa atgctacggc ttgagggcca tggggacccc ctacacctgg
     2401 aggaggtgaa gaggcacaag gtccacatct cagggacact gtggtggacc tatgtcatca
     2461 gcgtggtgtt ccggctgttg tttgaggccg tcttcatgta tgtcttttat ctgctctacc
     2521 ctggctatgc catggtgcgg ctggtcaagt gcgacgtcta cccctgcccc aacacagtgt
     2581 actgcttcgt gtcccgcccc accgagaaaa ccgtcttcac cgtcttcatg ctagctgcct
     2641 ctggcatctg catcatcctc aatgtggccg aggtggtgta cctcatcatc cgggcctgtg
     2701 cccgccgagc ccagcgccgc tccaatccac cttcccgcaa gggctcgggc ttcggccacc
     2761 gcctctcacc tgaatacaag cagaatgaga tcaacaagct gctgagtgag caggatggct
     2821 ccctgaaaga catactgcgc cgcagccctg gcaccggggc tgggctggct gaaaagagcg
     2881 accgctgctc ggcctgctag acgcgtcccc tctccctccc ccccccctaa cgttactggc
     2941 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     3001 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     3061 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     3121 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     3181 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     3241 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     3301 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     3361 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     3421 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     3481 tatggccaca accatgggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
     3541 tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctcg ccaccatggt
     3601 cgactcatca cgtcgtaagt ggaataagac aggtcacgca gtcagagcta taggtcggct
     3661 gagctcactc gagaacgtct atatcaaggc cgacaagcag aagaacggca tcaaggcgaa
     3721 cttccacatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc actaccagca
     3781 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcgtgca
     3841 gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3901 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta ccggagggag
     3961 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     4021 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg atgccaccta
     4081 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     4141 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     4201 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac atccaggagc gcaccatctt
     4261 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     4321 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     4381 caagctggag tacaacctgc cggaccaact gactgaagag cagatcgcag aatttaaaga
     4441 ggctttctcc ctatttgaca aggacgggga tgggacaata acaaccaagg agctggggac
     4501 ggtgatgcgg tctctggggc agaaccccac agaagcagag ctgcaggaca tgatcaatga
     4561 agtagatgcc gacggtgacg gcacaatcga cttccctgag ttcctgacaa tgatggcaag
     4621 aaaaatgaaa tacagggaca cggaagaaga aattagagaa gcgttcggtg tgtttgataa
     4681 ggatggcaat ggctacatca gtgcagcaga gcttcgccac gtgatgacaa accttggaga
     4741 gaagttaaca gatgaagagg ttgatgaaat gatcagggaa gcagacatcg atggggatgg
     4801 tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg aagtagggat ccggggttgg
     4861 ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg
     4921 gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca
     4981 gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc
     5041 ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg
     5101 cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac
     5161 cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc agcggccggg
     5221 aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc
     5281 ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc
     5341 gaatcaccga cctctctccc cagcaattca ccatgaccga gtacaagccc acggtgcgcc
     5401 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     5461 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     5521 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     5581 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     5641 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     5701 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     5761 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     5821 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     5881 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     5941 cctggtgcat gacccgcaag cccggtgccg aaggtagagg ttctctcctc acttgtggtg
     6001 atgttgaaga aaaccctggt ccaatgtcta gactggacaa gagcaaagtc ataaacggag
     6061 ctctggaatt actcaatggt gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa
     6121 agctgggagt tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg
     6181 atgccctgcc aatcgagatg ctggacaggc atcataccca cttctgcccc ctggaaggcg
     6241 agtcatggca agactttctg cggaacaacg ccaagtcata ccgctgtgct ctcctctcac
     6301 atcgcgacgg ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc
     6361 tggaaaatca gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg
     6421 ctctgtccgc cgtgggccac tttacactgg gctgcgtatt ggaggaacag gagcatcaag
     6481 tagcaaaaga ggaaagagag acacctacca ccgattctat gcccccactt ctgagacaag
     6541 caattgagct gttcgaccgg cagggagccg aacctgcctt ccttttcggc ctggaactaa
     6601 tcatatgtgg cctggagaaa cagctaaagt gcgaaagcgg cgggccgacc gacgcccttg
     6661 acgattttga cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc
     6721 tgcctgctga cgctcttgac gattttgacc ttgacatgct ccccgggtaa ctaagtaagg
     6781 atcgatccaa gatatcgtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     6841 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     6901 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     6961 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     7021 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     7081 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     7141 ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg
     7201 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     7261 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     7321 atctcccttt gggccgcctc cccgcctgtt tcgcctcggc gtccggacta gaggtacctt
     7381 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     7441 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     7501 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     7561 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     7621 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     7681 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     7741 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     7801 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     7861 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     7921 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     7981 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     8041 aggcctttca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     8101 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     8161 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     8221 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     8281 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     8341 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     8401 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     8461 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     8521 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     8581 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     8641 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     8701 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     8761 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     8821 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     8881 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     8941 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     9001 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     9061 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     9121 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     9181 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     9241 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     9301 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     9361 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     9421 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     9481 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     9541 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     9601 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     9661 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     9721 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     9781 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     9841 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     9901 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     9961 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
    10021 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
    10081 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
    10141 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
    10201 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
    10261 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
    10321 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
    10381 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa c
//
