LOCUS       sequence-307967-        9822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggybacTransposon-based tunable and temporal expression control of 
            Cas13d- eGFP and RUNX1 gRNA2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9822)
  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 9822)
  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-eGFP U6 RUNX1 g2
FEATURES             Location/Qualifiers
     source          1..9822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        34..245
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      265..274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             274..297
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             298..1041
                     /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             1051..1104
                     /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             1114..1713
                     /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(1114..1133)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1610..1630
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1734..1815
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1816..2064
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1816..1836
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1987..2006
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2066..2095
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     repeat_region   2301..2363
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2389..2405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2389..2405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2402..2424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2413..2429
                     /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)."
     promoter        complement(join(2437..2443,2444..2461,2462..2467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2482..2503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2620..2637)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2791..3379)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2871..2890)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3550..4341,4342..4410))
                     /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     4173..4192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4411..4515)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4583..4601
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4639..4661)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4761..4780
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4974..4996
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4988..5005
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4989..5005
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5016..5050
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5326..5460
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5679..5699)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(5700..6410,6411..6413,6414..6416))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(5700..6410,6411..6413,6414..6416))
                     /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(5742..5763)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6091..6110
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6350..6371
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(6417..6473)
                     /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             complement(6489..6536)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(6537..9434)
                     /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"
     promoter        complement(9447..9814)
                     /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    9572..9590
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9608..9626
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9644..9662
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9680..9698
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9716..9734
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9752..9770
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9788..9806
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aattccactc gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
       61 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
      121 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
      181 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
      241 cacaggtgtc gtgacgcggg atccgccacc atggattaca aagacgatga cgataagatg
      301 tctagactgg acaagagcaa agtcataaac tctgctctgg aattactcaa tggagtcggt
      361 atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg
      421 tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga gatgctggac
      481 aggcatcata cccactcctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac
      541 aacgccaagt cataccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc
      601 ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt
      661 cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca
      721 ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag agagacacct
      781 accaccgatt ctatgccccc acttctgaaa caagcaattg agctgttcga ccggcaggga
      841 gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta
      901 aagtgcgaaa gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga catgctccca
      961 gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt
     1021 gaccttgaca tgctccccgg gggcagcgga gagggcagag gttctcttct aacatgcggt
     1081 gacgtggagg agaatcccgg ccctaggctc gagatgaccg agtacaagcc cacggtgcgc
     1141 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     1201 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     1261 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     1321 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     1381 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     1441 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     1501 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     1561 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     1621 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     1681 acctggtgca tgacccgcaa gcccggtgcc tgatctagag ggcccgttta gttaacttgt
     1741 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     1801 catttttttc actgcgaggg cctatttccc atgattcctt catatttgca tatacgatac
     1861 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     1921 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     1981 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     2041 atcttgtgga aaggacgaaa caccgaaccc ctaccaactg gtcggggttt gaaactagtt
     2101 ttcatcattg ccagccattt ttttttaaaa gttttgttac tttatagaag aaattttgag
     2161 tttttgtttt tttttaataa ataaataaac ataaataaat tgtttgttga atttattatt
     2221 agtatgtaag tgtaaatata ataaaactta atatctattc aaattaataa ataaacctcg
     2281 atatacagac cgataaaaca catgcgtcaa ttttacgcat gattatcttt aacgtacgtc
     2341 acaatatgat tatctttcta gggttaagtc gaccttggcg taatcatggt catagctgtt
     2401 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2461 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2521 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2581 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     2641 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2701 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2761 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2821 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2881 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2941 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3001 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3061 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3121 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3181 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3241 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3301 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3361 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3421 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3481 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3541 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3601 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3661 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3721 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3781 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3841 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3901 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3961 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4021 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4081 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4141 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4201 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4261 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4321 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4381 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4441 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4501 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     4561 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     4621 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     4681 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4741 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     4801 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
     4861 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     4921 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     4981 acgacgttgt aaaacgacgg ccagtgaatt cttaacccta gaaagatagt ctgcgtaaaa
     5041 ttgacgcatg cattcttgaa atattgctct ctctttctaa atagcgcgaa tccgtcgctg
     5101 tgcatttagg acatctcagt cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg
     5161 taagtgtcac tgattttgaa ctataacgac cgcgtgagtc aaaatgacgc atgattatct
     5221 tttacgtgac ttttaagatt taactcatac gataattata ttgttatttc atgttctact
     5281 tacgtgataa cttattatat atatattttc ttgttataga tatcaaactt gtttattgca
     5341 gcttataatg gttacaaata aggcaatagc atcacaaatt tcacaaataa ggcatttttt
     5401 tcactgcatt ctagttttgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     5461 tcaaatccct cggaagctgc gcctgtctta ggttggagtg atacattttt atcactttta
     5521 cccgtctttg gattaggcag tagctctgac ggccctcctg tcttaggtta gtgaaaaatg
     5581 tcactctctt acccgtcatt ggctgtccag cttagctcgc aggggaggtg gtctactagt
     5641 gggttattga atatgatcgg aattcttact tgtacttata ccttacgctt cttctttggc
     5701 ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc
     5761 atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct caggtagtgg
     5821 ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg
     5881 agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt gatgccgttc
     5941 ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc cagcttgtgc
     6001 cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg
     6061 gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag
     6121 atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc
     6181 atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg
     6241 ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag
     6301 gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc
     6361 agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct caccattggc
     6421 ccgggattct cttcgacatc ccctgcttgt ttcaacaggg agaagttagt ggcgccgctg
     6481 ccgctagctt tcttcttctt agcctgtcca gccttctttg tggcggcagg tcgcttattg
     6541 ccggacacct tctttttctc cttgtcgaac ttggcggcct cgttcctatc gaacagggcc
     6601 tcgatgctca ggttcttaaa cctggggata cagtagccga aaggcacaca cagcagtttc
     6661 aggagcctat cgttgtactt cttctcgtca ttcacagcgt cgaagtactc ggacaccttt
     6721 ccgctgcttt tctcgtacct ctcattcatg ataattctct gcatgatgta atggtacagt
     6781 tggaagtagg aattgacctc ggcaatgtcg ttgatatagg cgtggacata cctggccact
     6841 tccaggtgga cggccttgtt tctgaacagg gtacatgtct tgttgtcaat tctcaggagg
     6901 tcctccctga tgatcacatt ccacttggtg ttcctcaggt aggcgttcag ggcggttttg
     6961 gccttctccc tgttaatctg cctggtgaac tcctcggctt tcttctcgtc gctgtatttg
     7021 atgtaattgg catacagctt ggggttggcg ctctcgaggc tgtcaatgct ctccttggct
     7081 ctttcagcca tctccttttc cacgtccttt ctcttatcgg gggcagtttc atcaatgcca
     7141 gcgcagagct tggtgacgga gctgaatccc ttctcttcca gtttcttgag attgatgtcg
     7201 tagcctttct ccttgtacag ttgagcatca cgctcgacgc aatggaatcc gatgacgtac
     7261 ctggcgttga tattgacaat attcttgagg atgtggtaga tcacggtgag gtacaggctg
     7321 atgatctttt taaacttctc cctctcggcg ttttccctgc cggtgtcctc aatgacgctc
     7381 cttttcttgt cgaattggtc gtagttcatt ccggtgatga tctttgtgag agcgtccacc
     7441 ttttcgctca cgctcttgcc cttatccttt ccgatacaag tttcgtagta cctgtcgatc
     7501 tggttcttgc cgttctggcc ctgttttttc tggatgtcag cgatcctgcc gagcacgaac
     7561 ttcaccacgg cctcgttttt ggcgatctca tggaggtggg caggatcacc gtatctgatc
     7621 aggtagtgga accttttatt gctgatcacg ttattaataa tgaaatttct catgccgtgc
     7681 ttgcctttct tgagcttgtt tccgttctcg tccagggaaa aggtgtcggc gagggccttg
     7741 agctcatcat aggacaggtt ggttcctaaa atacggatgg cgtcgatata catggccctc
     7801 ctggcatcgg caataggttc tcccattcta gcgaaggact tgatcagcct cagctcatcg
     7861 gcgatcttgg cggagtcttt gaaaaaggcg tattcctcca cgaacttagc gttgactccg
     7921 atgagaggca tcaccttcag gaagctctgg atgttatcga atttattaat cagggtggtc
     7981 aggaggtcgt tgatctcctt gccatccagg aacatggtca gggcatacat gagtttgctg
     8041 aaggcggaaa catcacggcc agcgggcagg attctgggca gtctaggggc gtccttcttc
     8101 ttatactctc ttgtcttgtt tcccctaaat tccttgatgt tgtgcatgat attttcgagc
     8161 tttctccaaa ttctattagc ttcatcgtag tagagggcat ccttctggtc gtcgttgaag
     8221 gagcccctca ggttaatcac aaagatatcc ttctcgctca gggacttctc attatcgggg
     8281 agggacttat tggcggcagc caccttggca tcctcttcga tgtaatacct ataaatcaca
     8341 aagtccatca tggtgtagac cttggtcctg atggagtcga acaccttatg atttttcctg
     8401 atctcggaca tatccttcct gtccagcatc acttccctga gcttggtgat attgaatccg
     8461 aggtttttct gctctttcat aatgctgaat ctgaaatatt gttcggcgaa ttcggcaggg
     8521 ttgattccca gagtttcggc aatatagttc acgttggcgg cggagttctt ggagaaggag
     8581 ttggtcagct cattggtgat cctgtcgtag aggtagttga gggtggagat gtattcgttg
     8641 tcgaggttct tatcgaggtt gtagagccag gtcctggaga tcctggactc ttcttcgttg
     8701 ttatggacca cccagtgcct cagtccgctc aggagggcca gaatgtcata gcattcgttg
     8761 ccgtaattga tgatgtaatt tctgccctcc ttgctgaaaa aggcctggcc gaaatagccg
     8821 agtctggggt tatcgaggaa gttgtcgaac tcgtcatact gggccttgat ggcgttgatg
     8881 agcttatcgt tattgttgaa agcggcccta tggtgctcgg ggtctttgaa ttcgtcgtag
     8941 gtatacactg tggagaactt gccgaatcca ataatgtcct tatccaggcc ggagatattg
     9001 ttgacggcgt aggcggcgtt ggtaatgtat tcggcgagga ttttttcaat gtccaggatg
     9061 ttatggatca cctggataca aatattgtca ttgccatcag cgctctcgcc gaagtacctc
     9121 ttttccagag tttccttcag gccgagcata tcctgctgga cgggtcctgt atacagaggg
     9181 ttgttagcca ccacggcgta gcccttagga tggctgaatt tggcgttgcc gatcttatag
     9241 ccggcgtttt tatcggccat ttcagcgctg aaggcctcgc cctcattcac gctcctgatg
     9301 ctgtcgccct ccacgatctt ttccagcctg gcgtcgctgc cttcggcgaa ggttgtcatg
     9361 tacactttgg agccggacac gagtgtggac ttcacgccca tgcccttggc gaaggacttt
     9421 tttttttcga tcatggtggc ggtaccttta cgagggtagg aagtggtacg gaaagttggt
     9481 ataagacaaa agtgttgtgg aattgaagtt tactcaaaaa atcagcactc ttttataggc
     9541 gccctggttt acataagcaa agcttatacg ttctctatca ctgataggga gtaaactgga
     9601 tatacgttct ctatcactga tagggagtaa actgtagata cgttctctat cactgatagg
     9661 gagtaaactg gtcatacgtt ctctatcact gatagggagt aaactcctta tacgttctct
     9721 atcactgata gggagtaaag tctgcatacg ttctctatca ctgataggga gtaaactctt
     9781 catacgttct ctatcactga tagggagtaa actcgaggtg at
//
