LOCUS       sequence-252519-       16735 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes pEF1a driving expression of TET3G P2A iRFP713, pEF1a 
            expressing sadCas9 fused to mRuby2 P2A mRuby2, and an sgRNA 
            targeting pUAS expressing mAzamiGreen in a PiggyBac destination 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16735)
  AUTHORS   Fonseca JP, Bonny AR, Kumar GR, Ng AH, Town J, Wu QC, Aslankoohi E, 
            Chen SY, Dods G, Harrigan P, Osimiri LC, Kistler AL, El-Samad H
  TITLE     A Toolkit for Rapid Modular Construction of Biological Circuits in 
            Mammalian Cells
  JOURNAL   ACS Synth Biol. 2019 Nov 15;8(11):2593-2606. doi: 
            10.1021/acssynbio.9b00322. Epub 2019 Nov 5.
  PUBMED    31686495
REFERENCE   2  (bases 1 to 16735)
  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"; doi: 
            "10.1021/acssynbio.9b00322"; journalName: "ACS Synth Biol"; date: 
            "2019-11-15- 15"; volume: "8"; issue: "11"; pages: "2593-2606"
COMMENT     Sequence Label: ARB365
FEATURES             Location/Qualifiers
     source          1..16735
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        540..743
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     694..714
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      765..774
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             771..1799
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             771..1799
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    1817..2024
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2231..2293
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(2344..2362)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(2402..3217)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2402..3217)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3125..3144
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3209..3228
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3442..4030
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3931..3950
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4142..4161
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4142..4160
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   4190..4224
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        4740..5918
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4971..5909
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     CDS             5929..6672
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             6688..6744
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6745..6765
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    7750..7974
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(8402..8435)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     8439..8459
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        8542..8617
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        8857..9068
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          9069..10007
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     CDS             10033..10053
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13234..13281
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             13288..13995
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     CDS             14011..14064
                     /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             14071..14091
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             14107..14814
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     primer_bind     complement(14852..14871)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     14906..14925
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             15447..15467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             15483..16154
                     /codon_start=1
                     /product="humanized monomeric Azami-Green fluorescent 
                     protein"
                     /label=hmAzami-Green
                     /note="mammalian codon-optimized"
                     /translation="VSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNVT
                     EGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTAT
                     SNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLLL
                     EGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLPS
                     QA"
     CDS             15483..16154
                     /codon_start=1
                     /product="monomeric Azami-Green fluorescent protein 
                     (Karasawa et al., 2003)"
                     /label=mAzami-Green
                     /translation="VSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNVT
                     EGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTAT
                     SNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLLL
                     EGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLPS
                     QA"
     primer_bind     complement(16251..16270)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    16316..16371
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(16370..16389)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
ORIGIN
        1 gttacagaga tgttacgaac cctgaacagg ttgactatta attgtgtctg cttgatgtgg
       61 acaccaggtg gcgctggaca tcagatttgg agaggcagtt gtctagggaa ccgggctctg
      121 tgccagcgca ggaggcaggc tggctctcct attccaggga tgctcatcca ggaaggaaag
      181 gttgcatgct ggacacacta accttacagc ggtgggctct atggggtacc tacgtaggta
      241 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      301 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      361 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      421 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      481 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      541 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
      601 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      661 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      721 gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatagccacc atgggtaaaa
      781 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtgt
      841 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
      901 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
      961 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     1021 ttagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     1081 tgcctgaaac cgaactgccc gctgttctgc aaccggtcgc ggaggccatg gatgcgatcg
     1141 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     1201 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     1261 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     1321 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     1381 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     1441 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     1501 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     1561 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     1621 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     1681 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     1741 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaataac
     1801 tcgagcgacg cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     1861 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     1921 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     1981 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2041 agtatcgata ctagttaaaa gttttgttac tttatagaag aaattttgag tttttgtttt
     2101 tttttaataa ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag
     2161 tgtaaatata ataaaactta atatctattc aaattaataa ataaacctcg atatacagac
     2221 cgataaaaca catgcgtcaa ttttacgcat gattatcttt aacgtacgtc acaatatgat
     2281 tatctttcta gggttaatct agtatacgcg tatgcggccg cttaattaat ccagcttttg
     2341 ttccctttag tgagggttaa ttccgagcgg ccgcgtgtta caaccaatta accaattctg
     2401 attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     2461 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     2521 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     2581 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     2641 ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc
     2701 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     2761 gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     2821 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     2881 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     2941 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3001 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3061 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     3121 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3181 gcctggagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     3241 tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc
     3301 agagattttg agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg
     3361 atcagtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3421 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3481 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3541 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3601 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3661 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3721 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3781 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3841 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3901 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3961 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4021 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4081 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgtg cggccgccaa
     4141 ttaatacgac tcactatagg gcgaattggg gcgcgccatt ctagattaac cctagaaaga
     4201 tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt ctaaatagcg
     4261 cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc cgtgaggcgt
     4321 gcttgtcaat gcggtaagtg tcactgattt tgaactataa cgaccgcgtg agtcaaaatg
     4381 acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat tatattgtta
     4441 tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta tagatatcaa
     4501 ctagaatgct agcatgggcc cagatctcca tgggccctga attcgcatct agactgaact
     4561 ggccgataat tgcagacgag gagcatcgcc cttccccggc cctcaggtaa gaggaccaaa
     4621 taccgtagcc gtttccaatt tcagtccttt agcgccacct ggtgctaact actctatcac
     4681 gcttttatcc aataactacc tttgtaaatt tcctttcaaa agttctggcc gggcgcggtg
     4741 gcaacggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     4801 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     4861 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     4921 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     4981 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     5041 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     5101 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     5161 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     5221 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     5281 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     5341 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     5401 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     5461 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     5521 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     5581 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     5641 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     5701 agttctcgcg cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     5761 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     5821 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     5881 tggttcaaag tttttttctt ccatttcagg tgtcgtgagc tagccactat gtctagactg
     5941 gacaagagca aagtcataaa ctctgctctg gaattactca atggagtcgg tatcgaaggc
     6001 ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc agcctaccct gtactggcac
     6061 gtgaagaaca agcgggccct gctcgatgcc ctgccaatcg agatgctgga caggcatcat
     6121 acccactcct gccccctgga aggcgagtca tggcaagact ttctgcggaa caacgccaag
     6181 tcataccgct gtgctctcct ctcacatcgc gacggggcta aagtgcatct cggcacccgc
     6241 ccaacagaga aacagtacga aaccctggaa aatcagctcg cgttcctgtg tcagcaaggc
     6301 ttctccctgg agaacgcact gtacgctctg tccgccgtgg gccactttac actgggctgc
     6361 gtattggagg aacaggagca tcaagtagca aaagaggaaa gagagacacc taccaccgat
     6421 tctatgcccc cacttctgaa acaagcaatt gagctgttcg accggcaggg agccgaacct
     6481 gccttccttt tcggcctgga actaatcata tgtggcctgg agaaacagct aaagtgcgaa
     6541 agcggcgggc cgaccgacgc ccttgacgat tttgacttag acatgctccc agccgatgcc
     6601 cttgacgact ttgaccttga tatgctgcct gctgacgctc ttgacgattt tgaccttgac
     6661 atgctccccg ggggatccgg aagcggagct actaacttca gcctgctgaa gcaggctggt
     6721 gacgtggagg agaaccctgg acctcccaag aagaagagga aggtggagga cgccggatcc
     6781 gctgaaggtt ctgttgctag acaaccagac ttgttgactt gtgacgacga accaatccac
     6841 atcccaggtg ctatccaacc acacggtttg ttgttggctt tggctgctga catgactatc
     6901 gttgctggtt ctgacaactt gccagaattg actggtttgg ctatcggtgc tttgatcggt
     6961 agatctgctg ctgacgtttt cgactctgaa actcacaaca gattgactat cgctttggct
     7021 gaaccaggtg ctgctgttgg tgctccaatc actgttggtt tcactatgag aaaggacgct
     7081 ggtttcatcg gttcttggca cagacacgac caattgatct tcttggaatt ggaaccacca
     7141 caaagagatg ttgctgaacc acaagctttc ttcagaagaa ctaactctgc tatcagaaga
     7201 ttgcaagctg ctgaaacttt ggaatctgct tgtgctgctg ctgctcaaga agttagaaag
     7261 atcactggtt tcgacagagt tatgatctac agattcgctt ctgacttctc tggtgaagtt
     7321 atcgctgaag acagatgtgc tgaagttgaa tctaagttgg gtttgcacta cccagcttct
     7381 actgttccag ctcaagctag aagattgtac actatcaacc cagttagaat catcccagac
     7441 atcaactaca gaccagttcc agttactcca gacttgaacc cagttactgg tagaccaatc
     7501 gacttgtctt tcgctatctt gagatctgtt tctccagttc acttggaatt catgagaaac
     7561 atcggtatgc acggtactat gtctatctct atcttgagag gtgaaagatt gtggggtttg
     7621 atcgtttgtc accacagaac tccatactac gttgacttgg acggtagaca agcttgtgaa
     7681 ttggttgctc aagttttggc ttggcaaatc ggtgttatgg aagaataact cgagtggccg
     7741 acgcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     7801 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     7861 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggtgcag gacagcaagg
     7921 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atgggctgcc
     7981 aaaccagatg tcaacacagc tacggtctgc cgcttcaggt ccatttacgg aagccgcaat
     8041 cctgcactat gaccaccagg gggagcgcta ggcccatccc tgcacaggta gctattttta
     8101 gaggttgctt ctagcctgga ggaagaaacg aaagaaataa aagactatga tacttttttt
     8161 ttttaaccgg accaacttat aaattatttg taagtccaat aaaaacggat ccgacgcgcc
     8221 atctctaggc ccgcgccggc cccctcgcac ggacttgtgg gagaagctcg gctactcccc
     8281 tgccccggtt aatttgcata taatatttcc tagtaactat agaggcttaa tgtgcgataa
     8341 aagacagata atctgttctt tttaatacta gctacatttt acatgatagg cttggatttc
     8401 tataacttcg tatagcatac attatacgaa gttataaaca gcacaaaagg aaactcaccc
     8461 taactgtaaa gtaattgtgt gttttgagac tataagtatc ccttggagaa ccaccttgtt
     8521 ggaacgtcgg agcactgtcc tgttatagta ctctggaaac agaatctact ataacaaggc
     8581 aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttttgctgga tgcacacact
     8641 ggcttaagat gacgggaccc tgtaaacgtg tccgccttgc cactttgtgc aatatttagc
     8701 tccttcagta gagggcgctg caggaacatg tcaggagggg gccttctctt ccggattctg
     8761 gattctggta tacacttgga gcgtggcatt tgggggacag ccactggtgc gcagccccat
     8821 caagttttgg tggcatccct aacggtgccc gtcagtgggc agagcgcaca tcgcccacag
     8881 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
     8941 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     9001 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     9061 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc
     9121 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc
     9181 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg
     9241 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc
     9301 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg
     9361 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta
     9421 tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg
     9481 cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc
     9541 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc
     9601 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag
     9661 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa
     9721 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc
     9781 cgtccaggca cctcgattag ttctcgcgct tttggagtac gtcgtcttta ggttgggggg
     9841 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag
     9901 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca
     9961 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgagcta
    10021 gccactatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccaag
    10081 cggaactaca tcctgggcct ggccatcggc atcaccagcg tgggctacgg catcatcgac
    10141 tacgagacac gggacgtgat cgatgccggc gtgcggctgt tcaaagaggc caacgtggaa
    10201 aacaacgagg gcaggcggag caagagaggc gccagaaggc tgaagcggcg gaggcggcat
    10261 agaatccaga gagtgaagaa gctgctgttc gactacaacc tgctgaccga ccacagcgag
    10321 ctgagcggca tcaaccccta cgaggccaga gtgaagggcc tgagccagaa gctgagcgag
    10381 gaagagttct ctgccgccct gctgcacctg gccaagagaa gaggcgtgca caacgtgaac
    10441 gaggtggaag aggacaccgg caacgagctg tccaccaaag agcagatcag ccggaacagc
    10501 aaggccctgg aagagaaata cgtggccgaa ctgcagctgg aacggctgaa gaaagacggc
    10561 gaagtgcggg gcagcatcaa cagattcaag accagcgact acgtgaaaga agccaaacag
    10621 ctgctgaagg tgcagaaggc ctaccaccag ctggaccaga gcttcatcga cacctacatc
    10681 gacctgctgg aaacccggcg gacctactat gagggacctg gcgagggcag ccccttcggc
    10741 tggaaggaca tcaaagaatg gtacgagatg ctgatgggcc actgcaccta cttccccgag
    10801 gaactgcgga gcgtgaagta cgcctacaac gccgacctgt acaacgccct gaacgacctg
    10861 aacaatctcg tgatcaccag ggacgagaac gagaagctgg aatattacga gaagttccag
    10921 atcatcgaga acgtgttcaa gcagaagaag aagcccaccc tgaagcagat cgccaaagaa
    10981 atcctcgtga acgaagagga tattaagggc tacagagtga ccagcaccgg caagcccgag
    11041 ttcaccaacc tgaaggtgta ccacgacatc aaggacatta ccgcccggaa agagattatt
    11101 gagaacgccg agctgctgga tcagattgcc aagatcctga ccatctacca gagcagcgag
    11161 gacatccagg aagaactgac caatctgaac tccgagctga cccaggaaga gatcgagcag
    11221 atctctaatc tgaagggcta taccggcacc cacaacctga gcctgaaggc catcaacctg
    11281 atcctggacg agctgtggca caccaacgac aaccagatcg ctatcttcaa ccggctgaag
    11341 ctggtgccca agaaggtgga cctgtcccag cagaaagaga tccccaccac cctggtggac
    11401 gacttcatcc tgagccccgt cgtgaagaga agcttcatcc agagcatcaa agtgatcaac
    11461 gccatcatca agaagtacgg cctgcccaac gacatcatta tcgagctggc ccgcgagaag
    11521 aactccaagg acgcccagaa aatgatcaac gagatgcaga agcggaaccg gcagaccaac
    11581 gagcggatcg aggaaatcat ccggaccacc ggcaaagaga acgccaagta cctgatcgag
    11641 aagatcaagc tgcacgacat gcaggaaggc aagtgcctgt acagcctgga agccatccct
    11701 ctggaagatc tgctgaacaa ccccttcaac tatgaggtgg accacatcat ccccagaagc
    11761 gtgtccttcg acaacagctt caacaacaag gtgctcgtga agcaggaaga agccagcaag
    11821 aagggcaacc ggaccccatt ccagtacctg agcagcagcg acagcaagat cagctacgaa
    11881 accttcaaga agcacatcct gaatctggcc aagggcaagg gcagaatcag caagaccaag
    11941 aaagagtatc tgctggaaga acgggacatc aacaggttct ccgtgcagaa agacttcatc
    12001 aaccggaacc tggtggatac cagatacgcc accagaggcc tgatgaacct gctgcggagc
    12061 tacttcagag tgaacaacct ggacgtgaaa gtgaagtcca tcaatggcgg cttcaccagc
    12121 tttctgcggc ggaagtggaa gtttaagaaa gagcggaaca aggggtacaa gcaccacgcc
    12181 gaggacgccc tgatcattgc caacgccgat ttcatcttca aagagtggaa gaaactggac
    12241 aaggccaaaa aagtgatgga aaaccagatg ttcgaggaaa agcaggccga gagcatgccc
    12301 gagatcgaaa ccgagcagga gtacaaagag atcttcatca ccccccacca gatcaagcac
    12361 attaaggact tcaaggacta caagtacagc caccgggtgg acaagaagcc taatagagag
    12421 ctgattaacg acaccctgta ctccacccgg aaggacgaca agggcaacac cctgatcgtg
    12481 aacaatctga acggcctgta cgacaaggac aatgacaagc tgaaaaagct gatcaacaag
    12541 agccccgaaa agctgctgat gtaccaccac gacccccaga cctaccagaa actgaagctg
    12601 attatggaac agtacggcga cgagaagaat cccctgtaca agtactacga ggaaaccggg
    12661 aactacctga ccaagtactc caaaaaggac aacggccccg tgatcaagaa gattaagtat
    12721 tacggcaaca aactgaacgc ccatctggac atcaccgacg actaccccaa cagcagaaac
    12781 aaggtcgtga agctgtccct gaagccctac agattcgacg tgtacctgga caatggcgtg
    12841 tacaagttcg tgaccgtgaa gaatctggat gtgatcaaaa aagaaaacta ctacgaagtg
    12901 aatagcaagt gctatgagga agctaagaag ctgaagaaga tcagcaacca ggccgagttt
    12961 atcgcctcct tctacaacaa cgatctgatc aagatcaacg gcgagctgta tagagtgatc
    13021 ggcgtgaaca acgacctgct gaaccggatc gaagtgaaca tgatcgacat cacctaccgc
    13081 gagtacctgg aaaacatgaa cgacaagagg ccccccagga tcattaagac aatcgcctcc
    13141 aagacccaga gcattaagaa gtacagcaca gacattctgg gcaacctgta tgaagtgaaa
    13201 tctaagaagc accctcagat catcaaaaag ggcaaaaggc cggcggccac gaaaaaggcc
    13261 ggccaggcaa aaaagaaaaa gggttctgtg tccaaaggag aggagttaat caaggaaaac
    13321 atgagaatga aagttgtcat ggagggctcc gttaatggtc accaattcaa gtgtacaggg
    13381 gaaggtgaag gtaatcctta catgggtaca caaactatga gaattaaagt aattgaaggc
    13441 ggaccactac catttgcatt tgacattctg gcaacgtcat tcatgtacgg atcacgaact
    13501 ttcatcaagt accctaaagg tataccagac tttttcaagc aatcttttcc agagggtttt
    13561 acatgggaaa gggttacaag atacgaagat gggggtgtcg tcacagttat gcaagatact
    13621 tcattagaag atggctgcct tgtctatcat gtgcaagtaa gaggggtgaa ttttccttct
    13681 aacggacctg tgatgcagaa aaagaccaaa ggttgggaac caaatactga aatgatgtac
    13741 ccagctgatg gaggtttgag aggctacaca cacatggcgc ttaaagttga tggtggaggt
    13801 catttgtctt gtagttttgt taccacttat cgttctaaaa agactgttgg caatatcaaa
    13861 atgccaggaa tacatgctgt agaccacaga ctagaaagac tcgaagagag cgataacgaa
    13921 atgttcgttg tacagagaga gcatgccgta gccaaatttg ctggcttagg cggtggtatg
    13981 gatgaattgt ataagggatc cggaagcgga gagggcagag gaagtctgct aacatgcggt
    14041 gacgtcgagg agaatcctgg acctggatcc cccaagaaga agaggaaggt ggaggacgcc
    14101 ggatccgtgt ccaaaggaga ggagttaatc aaggaaaaca tgagaatgaa agttgtcatg
    14161 gagggctccg ttaatggtca ccaattcaag tgtacagggg aaggtgaagg taatccttac
    14221 atgggtacac aaactatgag aattaaagta attgaaggcg gaccactacc atttgcattt
    14281 gacattctgg caacgtcatt catgtacgga tcacgaactt tcatcaagta ccctaaaggt
    14341 ataccagact ttttcaagca atcttttcca gagggtttta catgggaaag ggttacaaga
    14401 tacgaagatg ggggtgtcgt cacagttatg caagatactt cattagaaga tggctgcctt
    14461 gtctatcatg tgcaagtaag aggggtgaat tttccttcta acggacctgt gatgcagaaa
    14521 aagaccaaag gttgggaacc aaatactgaa atgatgtacc cagctgatgg aggtttgaga
    14581 ggctacacac acatggcgct taaagttgat ggtggaggtc atttgtcttg tagttttgtt
    14641 accacttatc gttctaaaaa gactgttggc aatatcaaaa tgccaggaat acatgctgta
    14701 gaccacagac tagaaagact cgaagagagc gataacgaaa tgttcgttgt acagagagag
    14761 catgccgtag ccaaatttgc tggcttaggc ggtggtatgg atgaattgta taagtaactc
    14821 gagtggcgat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
    14881 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
    14941 tataagctgc aataaacgct ggttctgtat gggcacagac aacctaacta atttcctggc
    15001 cctcaaggtg acttgcttga acttgccacc ttcgccacta gggggcagca ttggtttaca
    15061 cagggtgaac aaacgttcaa ttctaaacta atattctttg gtgggaaagt gtgttcattt
    15121 tggtttgttt tgaagaagtc acttcatacc tttgaactgg ggttaagctg gttaactccc
    15181 aaaaacggga gcactgtcct ccgaacgtcg gagcactgtc ctccgaacgt cggagcactg
    15241 tcctccgaac gtcggagcac tgtcctccga acggagcatg tcctccgaac gtcggagcac
    15301 tgtcctccga acgactagtt aggcgtgtac ggtgggaggc ctatataagc agagctcgtt
    15361 tagtgaaccg tcagatcgcc tgcagacgcc atccacgctg ttttgacctc catagaagac
    15421 accgggaccg atccagcgcc actatgccca agaagaagag gaaggtggag gacgccggtt
    15481 ctgtgagcgt gatcaagccc gagatgaaga tcaagctgtg catgaggggc accgtgaacg
    15541 gccacaactt cgtgatcgag ggcgagggca agggcaaccc ctacgagggc acccagatcc
    15601 tggacctgaa cgtgaccgag ggcgcccccc tgcccttcgc ctacgacatc ctgaccaccg
    15661 tgttccagta cggcaacagg gccttcacca agtaccccgc cgacatccag gactacttca
    15721 agcagacctt ccccgagggc taccactggg agaggagcat gacctacgag gaccagggca
    15781 tctgcaccgc caccagcaac atcagcatga ggggcgactg cttcttctac gacatcaggt
    15841 tcgacggcac caacttcccc cccaacggcc ccgtgatgca gaagaagacc ctgaagtggg
    15901 agcccagcac cgagaagatg tacgtggagg acggcgtgct gaagggcgac gtgaacatga
    15961 ggctgctgct ggagggcggc ggccactaca ggtgcgactt caagaccacc tacaaggcca
    16021 agaaggaggt gaggctgccc gacgcccaca agatcgacca caggatcgag atcctgaagc
    16081 acgacaagga ctacaacaag gtgaagctgt acgagaacgc cgtggccagg tactccatgc
    16141 tgcccagcca ggccggatcc taactcgaga ttcactcctc aggtgcaggc tgcctatcag
    16201 aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc
    16261 tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa
    16321 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
    16381 acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca
    16441 acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat
    16501 atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag
    16561 atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta
    16621 catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct
    16681 cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatgct gagca
//
