LOCUS       sequence-307945-        9512 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggybacTransposon-based tunable and temporal expression control of 
            Cas13d and nuclear eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9512)
  AUTHORS   Chang Y, Syahirah R, Oprescu SN, Wang X, Jung J, Cooper SH, 
            Torregrosa-Allen S, Elzey BD, Hsu AY, Randolph LN, Sun Y, Kuang S, 
            Broxmeyer HE, Deng Q, Lian X, Bao X
  TITLE     Chemically-defined generation of human hemogenic endothelium and 
            definitive hematopoietic progenitor cells.
  JOURNAL   Biomaterials. 2022 May 6;285:121569. doi: 
            10.1016/j.biomaterials.2022.121569.
  PUBMED    35567999
REFERENCE   2  (bases 1 to 9512)
  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: 
            "Biomaterials."; date: "2022-05-6"; volume: "285:121569. doi"; 
            pages: " 10.1016/j.biomaterials.2022.121569"
COMMENT     Sequence Label: XLone-Puro Cas13d P2A eGFP-NLS
FEATURES             Location/Qualifiers
     source          1..9512
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             389..3286
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS
                     IRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLE
                     KRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV
                     YTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYII
                     NYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITN
                     ELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRK
                     DMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFV
                     INLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPA
                     GRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAF
                     FKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDE
                     NGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ
                     KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRE
                     NAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLE
                     EKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKK
                     AEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVH
                     AYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCV
                     PFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGN"
     CDS             3287..3334
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3350..3406
                     /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             join(3407..3409,3410..3412,3413..4123)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3407..3409,3410..3412,3413..4123)
                     /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(3452..3473)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3713..3732)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4060..4081
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4124..4144
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4363..4497
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   4773..4807
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4818..4835)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4818..4834)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4827..4849)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5043..5062)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5162..5184
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5222..5240)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5308..5412
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5413..5481,5482..6273)
                     /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(5631..5650)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6444..7032
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6933..6952
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7186..7203
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7320..7341
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7356..7361,7362..7379,7380..7386)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7394..7410
                     /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     7399..7421
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7418..7434
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7418..7434
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   7460..7522
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    7698..7779
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(7800..8399)
                     /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(7883..7903)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     8380..8399
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(8409..8462)
                     /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             complement(8472..9215)
                     /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             complement(9216..9239)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      9239..9248
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(9268..9479)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta ccgccaccat gatcgaaaaa aaaaagtcct tcgccaaggg
      421 catgggcgtg aagtccacac tcgtgtccgg ctccaaagtg tacatgacaa ccttcgccga
      481 aggcagcgac gccaggctgg aaaagatcgt ggagggcgac agcatcagga gcgtgaatga
      541 gggcgaggcc ttcagcgctg aaatggccga taaaaacgcc ggctataaga tcggcaacgc
      601 caaattcagc catcctaagg gctacgccgt ggtggctaac aaccctctgt atacaggacc
      661 cgtccagcag gatatgctcg gcctgaagga aactctggaa aagaggtact tcggcgagag
      721 cgctgatggc aatgacaata tttgtatcca ggtgatccat aacatcctgg acattgaaaa
      781 aatcctcgcc gaatacatta ccaacgccgc ctacgccgtc aacaatatct ccggcctgga
      841 taaggacatt attggattcg gcaagttctc cacagtgtat acctacgacg aattcaaaga
      901 ccccgagcac catagggccg ctttcaacaa taacgataag ctcatcaacg ccatcaaggc
      961 ccagtatgac gagttcgaca acttcctcga taaccccaga ctcggctatt tcggccaggc
     1021 ctttttcagc aaggagggca gaaattacat catcaattac ggcaacgaat gctatgacat
     1081 tctggccctc ctgagcggac tgaggcactg ggtggtccat aacaacgaag aagagtccag
     1141 gatctccagg acctggctct acaacctcga taagaacctc gacaacgaat acatctccac
     1201 cctcaactac ctctacgaca ggatcaccaa tgagctgacc aactccttct ccaagaactc
     1261 cgccgccaac gtgaactata ttgccgaaac tctgggaatc aaccctgccg aattcgccga
     1321 acaatatttc agattcagca ttatgaaaga gcagaaaaac ctcggattca atatcaccaa
     1381 gctcagggaa gtgatgctgg acaggaagga tatgtccgag atcaggaaaa atcataaggt
     1441 gttcgactcc atcaggacca aggtctacac catgatggac tttgtgattt ataggtatta
     1501 catcgaagag gatgccaagg tggctgccgc caataagtcc ctccccgata atgagaagtc
     1561 cctgagcgag aaggatatct ttgtgattaa cctgaggggc tccttcaacg acgaccagaa
     1621 ggatgccctc tactacgatg aagctaatag aatttggaga aagctcgaaa atatcatgca
     1681 caacatcaag gaatttaggg gaaacaagac aagagagtat aagaagaagg acgcccctag
     1741 actgcccaga atcctgcccg ctggccgtga tgtttccgcc ttcagcaaac tcatgtatgc
     1801 cctgaccatg ttcctggatg gcaaggagat caacgacctc ctgaccaccc tgattaataa
     1861 attcgataac atccagagct tcctgaaggt gatgcctctc atcggagtca acgctaagtt
     1921 cgtggaggaa tacgcctttt tcaaagactc cgccaagatc gccgatgagc tgaggctgat
     1981 caagtccttc gctagaatgg gagaacctat tgccgatgcc aggagggcca tgtatatcga
     2041 cgccatccgt attttaggaa ccaacctgtc ctatgatgag ctcaaggccc tcgccgacac
     2101 cttttccctg gacgagaacg gaaacaagct caagaaaggc aagcacggca tgagaaattt
     2161 cattattaat aacgtgatca gcaataaaag gttccactac ctgatcagat acggtgatcc
     2221 tgcccacctc catgagatcg ccaaaaacga ggccgtggtg aagttcgtgc tcggcaggat
     2281 cgctgacatc cagaaaaaac agggccagaa cggcaagaac cagatcgaca ggtactacga
     2341 aacttgtatc ggaaaggata agggcaagag cgtgagcgaa aaggtggacg ctctcacaaa
     2401 gatcatcacc ggaatgaact acgaccaatt cgacaagaaa aggagcgtca ttgaggacac
     2461 cggcagggaa aacgccgaga gggagaagtt taaaaagatc atcagcctgt acctcaccgt
     2521 gatctaccac atcctcaaga atattgtcaa tatcaacgcc aggtacgtca tcggattcca
     2581 ttgcgtcgag cgtgatgctc aactgtacaa ggagaaaggc tacgacatca atctcaagaa
     2641 actggaagag aagggattca gctccgtcac caagctctgc gctggcattg atgaaactgc
     2701 ccccgataag agaaaggacg tggaaaagga gatggctgaa agagccaagg agagcattga
     2761 cagcctcgag agcgccaacc ccaagctgta tgccaattac atcaaataca gcgacgagaa
     2821 gaaagccgag gagttcacca ggcagattaa cagggagaag gccaaaaccg ccctgaacgc
     2881 ctacctgagg aacaccaagt ggaatgtgat catcagggag gacctcctga gaattgacaa
     2941 caagacatgt accctgttca gaaacaaggc cgtccacctg gaagtggcca ggtatgtcca
     3001 cgcctatatc aacgacattg ccgaggtcaa ttcctacttc caactgtacc attacatcat
     3061 gcagagaatt atcatgaatg agaggtacga gaaaagcagc ggaaaggtgt ccgagtactt
     3121 cgacgctgtg aatgacgaga agaagtacaa cgataggctc ctgaaactgc tgtgtgtgcc
     3181 tttcggctac tgtatcccca ggtttaagaa cctgagcatc gaggccctgt tcgataggaa
     3241 cgaggccgcc aagttcgaca aggagaaaaa gaaggtgtcc ggcaataagc gacctgccgc
     3301 cacaaagaag gctggacagg ctaagaagaa gaaagctagc ggcagcggcg ccactaactt
     3361 ctccctgttg aaacaagcag gggatgtcga agagaatccc gggccaatgg tgagcaaggg
     3421 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     3481 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     3541 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     3601 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     3661 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     3721 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     3781 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     3841 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     3901 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     3961 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     4021 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     4081 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagccaaaga agaagcgtaa
     4141 ggtataagta caagtaagaa ttccgatcat attcaataac ccactagtag accacctccc
     4201 ctgcgagcta agctggacag ccaatgacgg gtaagagagt gacatttttc actaacctaa
     4261 gacaggaggg ccgtcagagc tactgcctaa tccaaagacg ggtaaaagtg ataaaaatgt
     4321 atcactccaa cctaagacag gcgcagcttc cgagggattt gagatccaga catgataaga
     4381 tacattgatg agtttggaca aaccaaaact agaatgcagt gaaaaaaatg ccttatttgt
     4441 gaaatttgtg atgctattgc cttatttgta accattataa gctgcaataa acaagtttga
     4501 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
     4561 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
     4621 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
     4681 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
     4741 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
     4801 tctagggtta agaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     4861 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     4921 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     4981 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     5041 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5101 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5161 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5221 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5281 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5341 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5401 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5461 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5521 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5581 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5641 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5701 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5761 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5821 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5881 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5941 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6001 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6061 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6121 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6181 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6241 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6301 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6361 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6421 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6481 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6541 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6601 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6661 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6721 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6781 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6841 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6901 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6961 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7021 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7081 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     7141 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     7201 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     7261 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     7321 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     7381 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     7441 tacgccaagg tcgacttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa
     7501 tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat
     7561 ttgaatagat attaagtttt attatattta cacttacata ctaataataa attcaacaaa
     7621 caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata
     7681 aagtaacaaa acttttagca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     7741 gctttatttg taaccattat aagctgcaat aaacaagtta actaaacggg ccctctagat
     7801 caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac ctcgacgtcg
     7861 gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc ggaggtctcc
     7921 aggaaggcgg gcaccccggc gcgctcggcc gcctccactc cggggagcac gacggcgctg
     7981 cccagaccct tgccctggtg gtcgggcgag acgccgacgg tggccaggaa ccacgcgggc
     8041 tccttgggcc ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc cagccgggaa
     8101 ccgctcaact cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc ttcgacgctc
     8161 tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg cgacccacac cttgccgatg
     8221 tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc gatgtggcgg
     8281 tccggatcga cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc gagggtgcgt
     8341 acggccctgg ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta ctcggtcatc
     8401 tcgagcctag ggccgggatt ctcctccacg tcaccgcatg ttagaagact tcctctgccc
     8461 tctccgctgc ccccggggag catgtcaagg tcaaaatcgt caagagcgtc agcaggcagc
     8521 atatcaaggt caaagtcgtc aagggcatcg gctgggagca tgtctaagtc aaaatcgtca
     8581 agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt tctccaggcc acatatgatt
     8641 agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc ggtcgaacag ctcaattgct
     8701 tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct ctctttcctc ttttgctact
     8761 tgatgctcct gttcctccaa tacgcagccc agtgtaaagt ggcccacggc ggacagagcg
     8821 tacagtgcgt tctccaggga gaagccttgc tgacacagga acgcgagctg attttccagg
     8881 gtttcgtact gtttctctgt tgggcgggtg ccgagatgca ctttagcccc gtcgcgatgt
     8941 gagaggagag cacagcggta tgacttggcg ttgttccgca gaaagtcttg ccatgactcg
     9001 ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca tctcgattgg cagggcatcg
     9061 agcagggccc gcttgttctt cacgtgccag tacagggtag gctgctcaac tcccagcttt
     9121 tgagcgagtt tccttgtcgt caggccttcg ataccgactc cattgagtaa ttccagagca
     9181 gagtttatga ctttgctctt gtccagtcta gacatcttat cgtcatcgtc tttgtaatcc
     9241 atggtggcgg atcccgcgtc acgacacctg tgttctggcg gcaaacccgt tgcgaaaaag
     9301 aacgttcacg gcgactactg cacttatata cggttctccc ccaccctcgg gaaaaaggcg
     9361 gagccagtac acgacatcac tttcccagtt taccccgcgc caccttctct aggcaccggt
     9421 tcaattgccg acccctcccc ccaacttctc ggggactgtg ggcgatgtgc gctctgccca
     9481 ctgacgggca ccggagccac tcgagtggaa tt
//
