LOCUS       sequence-300187-        9385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggybacTransposon-based tunable and temporal expression control of 
            Cas13d and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9385)
  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 9385)
  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-BSD Cas13d P2A mCherry
FEATURES             Location/Qualifiers
     source          1..9385
                     /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             3410..3433
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3434..4141
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3576..3594)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3844..3863
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4347..4481
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   4757..4791
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4802..4819)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4802..4818)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4811..4833)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5027..5046)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5146..5168
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5206..5224)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5292..5396
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5397..5465,5466..6257)
                     /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(5615..5634)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6428..7016
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6917..6936
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7170..7187
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7304..7325
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7340..7345,7346..7363,7364..7370)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7378..7394
                     /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     7383..7405
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7402..7418
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7402..7418
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   7444..7506
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    7682..7763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(7880..8278)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(8279..8335)
                     /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(8345..9088)
                     /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(9089..9112)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      9112..9121
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(9141..9352)
                     /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 actacaagga
     3421 cgacgatgac aaggtgagca agggcgagga ggataacatg gccatcatca aggagttcat
     3481 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     3541 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     3601 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     3661 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     3721 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     3781 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     3841 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     3901 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     3961 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     4021 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     4081 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta
     4141 agaattccga tcatattcaa taacccacta gtagaccacc tcccctgcga gctaagctgg
     4201 acagccaatg acgggtaaga gagtgacatt tttcactaac ctaagacagg agggccgtca
     4261 gagctactgc ctaatccaaa gacgggtaaa agtgataaaa atgtatcact ccaacctaag
     4321 acaggcgcag cttccgaggg atttgagatc cagacatgat aagatacatt gatgagtttg
     4381 gacaaaccaa aactagaatg cagtgaaaaa aatgccttat ttgtgaaatt tgtgatgcta
     4441 ttgccttatt tgtaaccatt ataagctgca ataaacaagt ttgatatcta taacaagaaa
     4501 atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat
     4561 gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg
     4621 ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa
     4681 gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag
     4741 caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaagaatt
     4801 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4861 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     4921 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc
     4981 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     5041 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5101 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5161 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     5221 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     5281 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5341 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5401 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5461 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5521 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5581 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5641 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5701 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5761 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5821 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5881 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5941 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6001 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6061 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6121 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6181 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6241 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6301 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6361 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6421 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6481 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6541 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6601 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6661 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6721 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6781 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6841 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6901 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6961 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7021 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     7081 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     7141 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     7201 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     7261 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     7321 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     7381 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aaggtcgact
     7441 taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc gtaaaattga
     7501 cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag
     7561 ttttattata tttacactta catactaata ataaattcaa caaacaattt atttatgttt
     7621 atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt
     7681 agcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     7741 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     7801 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg
     7861 attatgatcc tctggagatt tagccctccc acacataacc agagggcagc aattcacgaa
     7921 tcccaactgc cgtcggctgt ccatcactgt ccttcactat ggctttgatc ccaggatgca
     7981 gatcgagaag cacctgtcgg caccgtccgc aggggctcaa gatgcccctg ttctcatttc
     8041 cgatcgcgac gatacaagtc aggttgccag ctgccgcagc agcagcagtg cccagcacca
     8101 cgagttctgc acaaggtccc ccagtaaaat gatatacatt gacaccagtg aagatgcggc
     8161 cgtcgctaga gagagctgcg ctggcgacgc tgtagtcttc agagatgggg atgctgttga
     8221 ttgtagccgt tgctctttca atgagggtgg attcttcttg agacaaaggc ttggccatgg
     8281 ggccggggtt ctcctccacg tcgccggcct gcttcagcag gctgaagttg gtggcgccgc
     8341 tgcccccggg gagcatgtca aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa
     8401 ggtcaaagtc gtcaagggca tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt
     8461 cggtcggccc gccgctttcg cactttagct gtttctccag gccacatatg attagttcca
     8521 ggccgaaaag gaaggcaggt tcggctccct gccggtcgaa cagctcaatt gcttgtttca
     8581 gaagtggggg catagaatcg gtggtaggtg tctctctttc ctcttttgct acttgatgct
     8641 cctgttcctc caatacgcag cccagtgtaa agtggcccac ggcggacaga gcgtacagtg
     8701 cgttctccag ggagaagcct tgctgacaca ggaacgcgag ctgattttcc agggtttcgt
     8761 actgtttctc tgttgggcgg gtgccgagat gcactttagc cccgtcgcga tgtgagagga
     8821 gagcacagcg gtatgacttg gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca
     8881 gggggcagga gtgggtatga tgcctgtcca gcatctcgat tggcagggca tcgagcaggg
     8941 cccgcttgtt cttcacgtgc cagtacaggg taggctgctc aactcccagc ttttgagcga
     9001 gtttccttgt cgtcaggcct tcgataccga ctccattgag taattccaga gcagagttta
     9061 tgactttgct cttgtccagt ctagacatct tatcgtcatc gtctttgtaa tccatggtgg
     9121 cggatcccgc gtcacgacac ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc
     9181 acggcgacta ctgcacttat atacggttct cccccaccct cgggaaaaag gcggagccag
     9241 tacacgacat cactttccca gtttaccccg cgccaccttc tctaggcacc ggttcaattg
     9301 ccgacccctc cccccaactt ctcggggact gtgggcgatg tgcgctctgc ccactgacgg
     9361 gcaccggagc cactcgagtg gaatt
//
