LOCUS       sequence-263327-       12473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a split firefly luciferase reporter based on the N 
            terminal carbohydrate recognition domain of human galectin 8, which 
            concentrates inside endosomes following endosomal disruption.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12473)
  AUTHORS   Kilchrist KV, Tierney JW, Duvall CL
  TITLE     Genetically encoded split-luciferase biosensors to measure endosome 
            disruption rapidly in live cells.
  JOURNAL   ACS Sens. 2020 Jun 23. doi: 10.1021/acssensors.0c00103.
  PUBMED    32573202
REFERENCE   2  (bases 1 to 12473)
  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: "ACS Sens. 
            2020 Jun 23. doi: 10.1021/acssensors.0c00103."
COMMENT     Sequence Label: pLenti NLuc398-G8NCRD IRES G8NCRD-CLuc394
FEATURES             Location/Qualifiers
     source          1..12473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    9..29
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        30..1208
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          261..1199
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1156..1176
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1208..1232
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1233..1242
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2955..2979)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(2980..3554,3555..3557,3558..3566)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3147..3164)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3374..3393
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     protein_bind    complement(4557..4577)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_feature    4590..5178
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4643..4663)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5061..5072)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     enhancer        5208..5587
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5588..5791
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5742..5762
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      5818..5827
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5824..5826,5827..5829,5830..6540)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5824..5826,5827..5829,5830..6540)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5869..5890)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6130..6149)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6477..6498
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6550..6603
                     /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             6604..7002
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             7074..7307
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7379..7500
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7416..7435)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7470..7489
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7540..7675
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7695..7714)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7696..7714)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7724..7741)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7724..7740)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7733..7755)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7882..8337
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7969..7988)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8179..8200
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8363..8467
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8468..8536,8537..9328)
                     /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(8686..8705)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9499..10087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9988..10007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10241..10258
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10375..10396
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10411..10416,10417..10434,10435..10441)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10449..10465
                     /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     10454..10476
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10473..10489
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10473..10489
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10508..10528
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10510..10528
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        10556..10782
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             10783..10963
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11010..11135
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11628..11861
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12046..12090
                     /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    12356..12473
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 actagtatca actttgtata gaaaagttgg gctccggtgc ccgtcagtgg gcagagcgca
       61 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
      121 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
      181 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
      241 ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta
      301 cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg
      361 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc
      421 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
      481 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt
      541 gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc
      601 tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc
      661 gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
      721 ctcaagctgg ccggcctgct ctggtgcctg gtctcgcgcc gccgtgtatc gccccgccct
      781 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
      841 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt
      901 cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg
      961 agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt
     1021 taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg
     1081 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg
     1141 gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
     1201 tgtcgtgaca agtttgtaca aaaaagcagg ctgccaccat ggaagatgcc aaaaacatta
     1261 agaagggccc agcgccattc tacccactcg aagacgggac cgccggcgag cagctgcaca
     1321 aagccatgaa gcgctacgcc ctggtgcccg gcaccatcgc ctttaccgac gcacatatcg
     1381 aggtggacat tacctacgcc gagtacttcg agatgagcgt tcggctggca gaagctatga
     1441 agcgctatgg gctgaataca aaccatcgga tcgtggtgtg cagcgagaat agcttgcagt
     1501 tcttcatgcc cgtgttgggt gccctgttca tcggtgtggc tgtggcccca gctaacgaca
     1561 tctacaacga gcgcgagctg ctgaacagca tgggcatcag ccagcccacc gtcgtattcg
     1621 tgagcaagaa agggctgcaa aagatcctca acgtgcaaaa gaagctaccg atcatacaaa
     1681 agatcatcat catggatagc aagaccgact accagggctt ccaaagcatg tacaccttcg
     1741 tgacttccca tttgccaccc ggcttcaacg agtacgactt cgtgcccgag agcttcgacc
     1801 gggacaaaac catcgccctg atcatgaaca gtagtggcag taccggattg cccaagggcg
     1861 tagccctacc gcaccgcacc gcttgtgtcc gattcagtca tgcccgcgac cccatcttcg
     1921 gcaaccagat catccccgac accgctatcc tcagcgtggt gccatttcac cacggcttcg
     1981 gcatgttcac cacgctgggc tacttgatct gcggctttcg ggtcgtgctc atgtaccgct
     2041 tcgaggagga gctattcttg cgcagcttgc aagactataa gattcaatct gccctgctgg
     2101 tgcccacact atttagcttc ttcgctaaga gcactctcat cgacaagtac gacctaagca
     2161 acttgcacga gatcgccagc ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg
     2221 ccaaacgctt ccacctacca ggcatccgcc agggctacgg cctgacagaa acaaccagcg
     2281 ccattctgat cacccccgaa ggggacgaca agcctggcgc agtaggcaag gtggtgccct
     2341 tcttcgaggc taaggtggtg gacttggaca ccggtaagac actgggtgtg aaccagcgcg
     2401 gcgagctgtg cgtccgtggc cccatgatca tgtctggtgg cggaggctcg ggcggaggtg
     2461 ggtcgggtgg cggcggatca atgatgttgt ccttaaacaa cctacagaat atcatctata
     2521 acccggtaat cccgtttgtt ggcaccattc ctgatcagct ggatcctgga actttgattg
     2581 tgatacgtgg gcatgttcct agtgacgcag acagattcca ggtggatctg cagaatggca
     2641 gcagcatgaa acctcgagcc gatgtggcct ttcatttcaa tcctcgtttc aaaagggccg
     2701 gctgcattgt ttgcaatact ttgataaatg aaaaatgggg acgggaagag atcacctatg
     2761 acacgccttt caaaagagaa aagtcttttg agatcgtgat tatggtgctg aaggacaaat
     2821 tccaggtggc tgtaaatgga aaacatactc tgctctatgg ccacaggatc ggcccagaga
     2881 aaatagacac tctgggcatt tatggcaaag tgaatattca ctcaattggt tttagcttca
     2941 gctcggacta atagacccag ctttcttgta caaagtgggc ccctctccct cccccccccc
     3001 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     3061 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     3121 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     3181 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     3241 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     3301 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     3361 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     3421 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     3481 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
     3541 aacacgatga taatatggcc acaaccatga tgttgtcctt aaacaaccta cagaatatca
     3601 tctataaccc ggtaatcccg tttgttggca ccattcctga tcagctggat cctggaactt
     3661 tgattgtgat acgtgggcat gttcctagtg acgcagacag attccaggtg gatctgcaga
     3721 atggcagcag catgaaacct cgagccgatg tggcctttca tttcaatcct cgtttcaaaa
     3781 gggccggctg cattgtttgc aatactttga taaatgaaaa atggggacgg gaagagatca
     3841 cctatgacac gcctttcaaa agagaaaagt cttttgagat cgtgattatg gtgctgaagg
     3901 acaaattcca ggtggctgta aatggaaaac atactctgct ctatggccac aggatcggcc
     3961 cagagaaaat agacactctg ggcatttatg gcaaagtgaa tattcactca attggtttta
     4021 gcttcagctc ggactctggt ggcggaggct cgggcggagg tgggtcgggt ggcggcggat
     4081 cacccatgat catgagcggc tacgttaaca accccgaggc tacaaacgct ctcatcgaca
     4141 aggacggctg gctgcacagc ggcgacatcg cctactggga cgaggacgag cacttcttca
     4201 tcgtggaccg gctgaagagc ctgatcaaat acaagggcta ccaggtagcc ccagccgaac
     4261 tggagagcat cctgctgcaa caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg
     4321 acgacgatgc cggcgagctg cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga
     4381 ccgagaagga gatcgtggac tatgtggcca gccaggttac aaccgccaag aagctgcgcg
     4441 gtggtgttgt gttcgtggac gaggtgccta aaggactgac cggcaagttg gacgcccgca
     4501 agatccgcga gattctcatt aaggccaaga agggcggcaa gatcgccgtg taatagcaac
     4561 tttattatac atagttgatc aattccgata atcaacctct ggattacaaa atttgtgaaa
     4621 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4681 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4741 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4801 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4861 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     4921 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     4981 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     5041 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     5101 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     5161 tttgggccgc ctccccgcat cgggaattcc cgcggttcga acgcgttgac attgattatt
     5221 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     5281 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     5341 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     5401 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     5461 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     5521 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     5581 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     5641 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     5701 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     5761 tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgcgcc
     5821 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     5881 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     5941 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     6001 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     6061 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     6121 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     6181 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     6241 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     6301 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     6361 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     6421 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     6481 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     6541 ggctccggag agggcagggg aagtcttcta acatgcgggg acgtggagga aaatcccggc
     6601 cccatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
     6661 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
     6721 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
     6781 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
     6841 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
     6901 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
     6961 attcgtgaat tgctgccctc tggttatgtg tgggagggct aaggtacctt taagaccaat
     7021 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     7081 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
     7141 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     7201 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     7261 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg
     7321 tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa
     7381 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     7441 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     7501 tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc
     7561 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     7621 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg
     7681 acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     7741 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     7801 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     7861 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     7921 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     7981 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     8041 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     8101 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     8161 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     8221 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     8281 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     8341 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     8401 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     8461 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     8521 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     8581 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     8641 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     8701 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     8761 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     8821 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     8881 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     8941 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     9001 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     9061 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     9121 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     9181 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     9241 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     9301 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     9361 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     9421 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     9481 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     9541 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     9601 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     9661 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     9721 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     9781 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     9841 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     9901 cgccacgctt cccgaagaga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     9961 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
    10021 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
    10081 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
    10141 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
    10201 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
    10261 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
    10321 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
    10381 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
    10441 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
    10501 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag
    10561 tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta
    10621 caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc
    10681 ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat
    10741 tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac
    10801 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
    10861 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
    10921 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
    10981 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
    11041 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
    11101 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
    11161 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
    11221 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
    11281 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
    11341 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
    11401 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
    11461 ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
    11521 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
    11581 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
    11641 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
    11701 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
    11761 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
    11821 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
    11881 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
    11941 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
    12001 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
    12061 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
    12121 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
    12181 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
    12241 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
    12301 agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg ctagctttta
    12361 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
    12421 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat ttt
//
