LOCUS       sequence-307954-        9817 bp    DNA     circular SYN 14-APR-2025
DEFINITION  PiggybacTransposon-based tunable and temporal expression control of 
            Cas13d- eGFP and U6 gRNA backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9817)
  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 9817)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 BbsI
FEATURES             Location/Qualifiers
     source          1..9817
                     /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   2296..2358
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2384..2400)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2384..2400)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2397..2419)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2408..2424
                     /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(2432..2438,2439..2456,2457..2462))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2477..2498
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2615..2632)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2786..3374)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2866..2885)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3545..4336,4337..4405))
                     /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     4168..4187
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4406..4510)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4578..4596
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4634..4656)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4756..4775
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4969..4991
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4983..5000
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4984..5000
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5011..5045
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5321..5455
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5674..5694)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(5695..6405,6406..6408,6409..6411))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(5695..6405,6406..6408,6409..6411))
                     /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(5737..5758)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6086..6105
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6345..6366
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(6412..6468)
                     /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(6484..6531)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(6532..9429)
                     /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(9442..9809)
                     /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    9567..9585
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9603..9621
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9639..9657
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9675..9693
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9711..9729
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9747..9765
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    9783..9801
                     /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 gaaacgggtc
     2101 ttcgagaaga cctttttttt taaaagtttt gttactttat agaagaaatt ttgagttttt
     2161 gttttttttt aataaataaa taaacataaa taaattgttt gttgaattta ttattagtat
     2221 gtaagtgtaa atataataaa acttaatatc tattcaaatt aataaataaa cctcgatata
     2281 cagaccgata aaacacatgc gtcaatttta cgcatgatta tctttaacgt acgtcacaat
     2341 atgattatct ttctagggtt aagtcgacct tggcgtaatc atggtcatag ctgtttcctg
     2401 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     2461 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     2521 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2581 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2641 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2701 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2761 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2821 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2881 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2941 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3001 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3061 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3121 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3181 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3241 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3301 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3361 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3421 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3481 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3541 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3601 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3661 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3721 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3781 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3841 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3901 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3961 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4021 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4081 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4141 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4201 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4261 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4321 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4381 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4441 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4501 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     4561 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     4621 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     4681 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     4741 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     4801 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc
     4861 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     4921 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     4981 gttgtaaaac gacggccagt gaattcttaa ccctagaaag atagtctgcg taaaattgac
     5041 gcatgcattc ttgaaatatt gctctctctt tctaaatagc gcgaatccgt cgctgtgcat
     5101 ttaggacatc tcagtcgccg cttggagctc ccgtgaggcg tgcttgtcaa tgcggtaagt
     5161 gtcactgatt ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac
     5221 gtgactttta agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt
     5281 gataacttat tatatatata ttttcttgtt atagatatca aacttgttta ttgcagctta
     5341 taatggttac aaataaggca atagcatcac aaatttcaca aataaggcat ttttttcact
     5401 gcattctagt tttggtttgt ccaaactcat caatgtatct tatcatgtct ggatctcaaa
     5461 tccctcggaa gctgcgcctg tcttaggttg gagtgataca tttttatcac ttttacccgt
     5521 ctttggatta ggcagtagct ctgacggccc tcctgtctta ggttagtgaa aaatgtcact
     5581 ctcttacccg tcattggctg tccagcttag ctcgcagggg aggtggtcta ctagtgggtt
     5641 attgaatatg atcggaattc ttacttgtac ttatacctta cgcttcttct ttggcttgta
     5701 cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg
     5761 atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc
     5821 gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg
     5881 cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg
     5941 cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag
     6001 gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc
     6061 gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt
     6121 gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg
     6181 gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca
     6241 gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc
     6301 atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc
     6361 gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca ttggcccggg
     6421 attctcttcg acatcccctg cttgtttcaa cagggagaag ttagtggcgc cgctgccgct
     6481 agctttcttc ttcttagcct gtccagcctt ctttgtggcg gcaggtcgct tattgccgga
     6541 caccttcttt ttctccttgt cgaacttggc ggcctcgttc ctatcgaaca gggcctcgat
     6601 gctcaggttc ttaaacctgg ggatacagta gccgaaaggc acacacagca gtttcaggag
     6661 cctatcgttg tacttcttct cgtcattcac agcgtcgaag tactcggaca cctttccgct
     6721 gcttttctcg tacctctcat tcatgataat tctctgcatg atgtaatggt acagttggaa
     6781 gtaggaattg acctcggcaa tgtcgttgat ataggcgtgg acatacctgg ccacttccag
     6841 gtggacggcc ttgtttctga acagggtaca tgtcttgttg tcaattctca ggaggtcctc
     6901 cctgatgatc acattccact tggtgttcct caggtaggcg ttcagggcgg ttttggcctt
     6961 ctccctgtta atctgcctgg tgaactcctc ggctttcttc tcgtcgctgt atttgatgta
     7021 attggcatac agcttggggt tggcgctctc gaggctgtca atgctctcct tggctctttc
     7081 agccatctcc ttttccacgt cctttctctt atcgggggca gtttcatcaa tgccagcgca
     7141 gagcttggtg acggagctga atcccttctc ttccagtttc ttgagattga tgtcgtagcc
     7201 tttctccttg tacagttgag catcacgctc gacgcaatgg aatccgatga cgtacctggc
     7261 gttgatattg acaatattct tgaggatgtg gtagatcacg gtgaggtaca ggctgatgat
     7321 ctttttaaac ttctccctct cggcgttttc cctgccggtg tcctcaatga cgctcctttt
     7381 cttgtcgaat tggtcgtagt tcattccggt gatgatcttt gtgagagcgt ccaccttttc
     7441 gctcacgctc ttgcccttat cctttccgat acaagtttcg tagtacctgt cgatctggtt
     7501 cttgccgttc tggccctgtt ttttctggat gtcagcgatc ctgccgagca cgaacttcac
     7561 cacggcctcg tttttggcga tctcatggag gtgggcagga tcaccgtatc tgatcaggta
     7621 gtggaacctt ttattgctga tcacgttatt aataatgaaa tttctcatgc cgtgcttgcc
     7681 tttcttgagc ttgtttccgt tctcgtccag ggaaaaggtg tcggcgaggg ccttgagctc
     7741 atcataggac aggttggttc ctaaaatacg gatggcgtcg atatacatgg ccctcctggc
     7801 atcggcaata ggttctccca ttctagcgaa ggacttgatc agcctcagct catcggcgat
     7861 cttggcggag tctttgaaaa aggcgtattc ctccacgaac ttagcgttga ctccgatgag
     7921 aggcatcacc ttcaggaagc tctggatgtt atcgaattta ttaatcaggg tggtcaggag
     7981 gtcgttgatc tccttgccat ccaggaacat ggtcagggca tacatgagtt tgctgaaggc
     8041 ggaaacatca cggccagcgg gcaggattct gggcagtcta ggggcgtcct tcttcttata
     8101 ctctcttgtc ttgtttcccc taaattcctt gatgttgtgc atgatatttt cgagctttct
     8161 ccaaattcta ttagcttcat cgtagtagag ggcatccttc tggtcgtcgt tgaaggagcc
     8221 cctcaggtta atcacaaaga tatccttctc gctcagggac ttctcattat cggggaggga
     8281 cttattggcg gcagccacct tggcatcctc ttcgatgtaa tacctataaa tcacaaagtc
     8341 catcatggtg tagaccttgg tcctgatgga gtcgaacacc ttatgatttt tcctgatctc
     8401 ggacatatcc ttcctgtcca gcatcacttc cctgagcttg gtgatattga atccgaggtt
     8461 tttctgctct ttcataatgc tgaatctgaa atattgttcg gcgaattcgg cagggttgat
     8521 tcccagagtt tcggcaatat agttcacgtt ggcggcggag ttcttggaga aggagttggt
     8581 cagctcattg gtgatcctgt cgtagaggta gttgagggtg gagatgtatt cgttgtcgag
     8641 gttcttatcg aggttgtaga gccaggtcct ggagatcctg gactcttctt cgttgttatg
     8701 gaccacccag tgcctcagtc cgctcaggag ggccagaatg tcatagcatt cgttgccgta
     8761 attgatgatg taatttctgc cctccttgct gaaaaaggcc tggccgaaat agccgagtct
     8821 ggggttatcg aggaagttgt cgaactcgtc atactgggcc ttgatggcgt tgatgagctt
     8881 atcgttattg ttgaaagcgg ccctatggtg ctcggggtct ttgaattcgt cgtaggtata
     8941 cactgtggag aacttgccga atccaataat gtccttatcc aggccggaga tattgttgac
     9001 ggcgtaggcg gcgttggtaa tgtattcggc gaggattttt tcaatgtcca ggatgttatg
     9061 gatcacctgg atacaaatat tgtcattgcc atcagcgctc tcgccgaagt acctcttttc
     9121 cagagtttcc ttcaggccga gcatatcctg ctggacgggt cctgtataca gagggttgtt
     9181 agccaccacg gcgtagccct taggatggct gaatttggcg ttgccgatct tatagccggc
     9241 gtttttatcg gccatttcag cgctgaaggc ctcgccctca ttcacgctcc tgatgctgtc
     9301 gccctccacg atcttttcca gcctggcgtc gctgccttcg gcgaaggttg tcatgtacac
     9361 tttggagccg gacacgagtg tggacttcac gcccatgccc ttggcgaagg actttttttt
     9421 ttcgatcatg gtggcggtac ctttacgagg gtaggaagtg gtacggaaag ttggtataag
     9481 acaaaagtgt tgtggaattg aagtttactc aaaaaatcag cactctttta taggcgccct
     9541 ggtttacata agcaaagctt atacgttctc tatcactgat agggagtaaa ctggatatac
     9601 gttctctatc actgataggg agtaaactgt agatacgttc tctatcactg atagggagta
     9661 aactggtcat acgttctcta tcactgatag ggagtaaact ccttatacgt tctctatcac
     9721 tgatagggag taaagtctgc atacgttctc tatcactgat agggagtaaa ctcttcatac
     9781 gttctctatc actgataggg agtaaactcg aggtgat
//
