LOCUS       sequence-450448-       13808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dox-inducible ABE8.20-NG for mammalian cells. piggyBac 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13808)
  AUTHORS   Osgood NRB, Zawalick NM, Sawyer CB, Cowan QT, Gu S, Mawson SJ, 
            Ranzau BL, Li L, Gymrek M, Goren A, Komor AC
  TITLE     Genome editing with programmable base editors in human cells
  JOURNAL   Methods in Enzymology, February 5, 2025
REFERENCE   2  (bases 1 to 13808)
  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: "Methods in 
            Enzymology, February 5, 2025"
COMMENT     Sequence Label: piggyBac-ABE8.20-NG
FEATURES             Location/Qualifiers
     source          1..13808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(14..469)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(151..172)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     363..382
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     596..618
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     610..627
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     611..627
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   757..819
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1268..1646
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1276..1294
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1312..1330
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1348..1366
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1384..1402
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1420..1438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1456..1474
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1492..1510
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1555..1579
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1682..2179
                     /codon_start=1
                     /product="DNA adenosine deaminase evolved from the E. coli 
                     tRNA adenosine deaminase TadA (Gaudelli et al., 2017)"
                     /label=TadA*(7.10)
                     /translation="SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWN
                     RAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRVVFG
                     VRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQS
                     STD"
     CDS             2276..6376
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6419..6439
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6449..6505
                     /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             6506..7213
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6651..6669)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6919..6938
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             7226..7246
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7255..7272
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7295..7312)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7301..7525
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        7786..7997
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             8010..8278
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             8312..9055
                     /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             9056..9109
                     /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             9131..10150
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             9131..10150
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(10207..10227)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    10261..10382
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10298..10317)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10352..10371
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   11145..11179
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11670..11686)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11670..11686)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11683..11705)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11694..11710
                     /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(11718..11724,11725..11742,11743..11748))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11763..11784
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11901..11918)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12072..12660)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12152..12171)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12831..13622,13623..13691))
                     /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     13454..13473
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13692..13796)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
       61 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
      121 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
      181 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
      241 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
      301 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
      361 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
      421 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
      481 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
      541 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
      601 tcacgacgtt gtaaaacgac ggccagtgag cgcgcctcgt tcattcacgt ttttgaaccc
      661 gtggaggacg ggcagactcg cggtgcaaat gtgttttaca gcgtgatgga gcagatgaag
      721 atgctcgaca cgctgcagaa cacgcagcta gattaaccct agaaagataa tcatattgtg
      781 acgtacgtta aagataatca tgtgtaaaat tgacgcatgt gttttatcgg tctgtatatc
      841 gaggtttatt tattaatttg aatagatatt aagttttatt atatttacac ttacatacta
      901 ataataaatt caacaaacaa tttatttatg tttatttatt tattaaaaaa aacaaaaact
      961 caaaatttct tctataaagt aacaaaactt ttatgaggga cagccccccc ccaaagcccc
     1021 cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg
     1081 ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca
     1141 cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc
     1201 agacacctgg ggggatacgg ggaaaaggcc tccacggcca ctagtctttc gtcttcaaga
     1261 attcctcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
     1321 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
     1381 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
     1441 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
     1501 gtgatagaga acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg
     1561 tttagtgaac cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt
     1621 tccgtaccac ttcctaccct cgtaaaggtc tagagctagc gaattcgaat ttgccaccat
     1681 gtccgaagtc gagttttccc atgagtactg gatgagacac gcattgactc tcgcaaagag
     1741 ggctcgagat gaacgcgagg tgcccgtggg ggcagtactc gtgctcaaca atcgcgtaat
     1801 cggcgaaggt tggaataggg caatcggact ccacgacccc actgcacatg cggaaatcat
     1861 ggcccttcga cagggagggc ttgtgatgca gaattatcga ctttatgatg cgacgctgta
     1921 ctcgacgttt gaaccttgcg taatgtgcgc gggagctatg attcactccc gcattggacg
     1981 agttgtattc ggtgttcgca acgccaagac gggtgccgca ggttcactga tggacgtgct
     2041 gcatcatcca ggcatgaacc accgggtaga aatcacagaa ggcatattgg cggacgaatg
     2101 tgcggcgctg ttgtgtcgtt tttttcgcat gcccaggcgg gtctttaacg cccagaaaaa
     2161 agcacaatcc tctactgact ctggtggttc ttctggtggt tctagcggca gcgagactcc
     2221 cgggacctca gagtccgcca cacccgaaag ttctggtggt tcttctggtg gttctgacaa
     2281 gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga
     2341 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     2401 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     2461 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     2521 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     2581 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     2641 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     2701 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     2761 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     2821 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     2881 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     2941 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     3001 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     3061 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     3121 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     3181 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     3241 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     3301 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     3361 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     3421 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     3481 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     3541 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     3601 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     3661 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     3721 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     3781 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     3841 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     3901 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     3961 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     4021 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     4081 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     4141 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     4201 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     4261 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     4321 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     4381 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     4441 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     4501 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     4561 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     4621 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     4681 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     4741 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     4801 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     4861 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     4921 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     4981 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     5041 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     5101 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     5161 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     5221 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     5281 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     5341 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     5401 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     5461 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     5521 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     5581 cggcttcagc aaagagtcta tccggcccaa gaggaacagc gataagctga tcgccagaaa
     5641 gaaggactgg gaccctaaga agtacggcgg cttcgtgagc cccaccgtgg cctattctgt
     5701 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     5761 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     5821 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     5881 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gcccggttcc tgcagaaggg
     5941 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     6001 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     6061 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     6121 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     6181 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctagggc
     6241 cttcaagtac tttgacacca ccatcgaccg gaaggtgtac cgtagcacca aagaggtgct
     6301 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     6361 tcagctggga ggtgactctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc
     6421 caagaagaag aggaaagtcg gaagcggagc tactaacttc agcctgctga agcaggctgg
     6481 agacgtggag gagaaccctg gacctatggt gagcaagggc gaggaggata acatggccat
     6541 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     6601 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     6661 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     6721 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     6781 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     6841 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     6901 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc
     6961 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     7021 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     7081 gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga
     7141 ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga
     7201 cgagctgtac aagtctggtg gttctcccaa gaagaagagg aaagtctaac cggtcatcat
     7261 caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     7321 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     7381 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     7441 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     7501 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctataaa
     7561 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     7621 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     7681 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg aaatcggatc
     7741 cagctaaatc taagtggatc tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat
     7801 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt gcctagagaa
     7861 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     7921 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     7981 ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca cgcgcccgcc
     8041 gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg
     8101 gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg agaccgggcc
     8161 tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg ctttgcctga
     8221 ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt tacagatcta
     8281 agctgtgacc ggcgcctact ctagagccgc catgtctaga ctggacaaga gcaaagtcat
     8341 aaactctgct ctggaattac tcaatggagt cggtatcgaa ggcctgacga caaggaaact
     8401 cgctcaaaag ctgggagttg agcagcctac cctgtactgg cacgtgaaga acaagcgggc
     8461 cctgctcgat gccctgccaa tcgagatgct ggacaggcat catacccact cctgccccct
     8521 ggaaggcgag tcatggcaag actttctgcg gaacaacgcc aagtcatacc gctgtgctct
     8581 cctctcacat cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag agaaacagta
     8641 cgaaaccctg gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc
     8701 actgtacgct ctgtccgccg tgggccactt tacactgggc tgcgtattgg aggaacagga
     8761 gcatcaagta gcaaaagagg aaagagagac acctaccacc gattctatgc ccccacttct
     8821 gaaacaagca attgagctgt tcgaccggca gggagccgaa cctgccttcc ttttcggcct
     8881 ggaactaatc atatgtggcc tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga
     8941 cgcccttgac gattttgact tagacatgct cccagccgat gcccttgacg actttgacct
     9001 tgatatgctg cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccggggaggg
     9061 cagaggaagt cttctaacat gcggtgacgt ggaggagaat cccggccctg aattcatgga
     9121 tagatccgga aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
     9181 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
     9241 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
     9301 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
     9361 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
     9421 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
     9481 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     9541 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
     9601 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     9661 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     9721 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     9781 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     9841 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     9901 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     9961 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
    10021 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
    10081 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
    10141 aaaggaataa gccaaggaac ctctggatta caaaatttgt gaaagattga ctggtattct
    10201 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcagtt
    10261 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
    10321 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
    10381 tatcatgtct ggaattgact caaatgatgt caattagtct atcagaagct catctggtct
    10441 cccttccggg ggacaagaca tccctgttta atatttaaac agcagtgttc ccaaactggg
    10501 ttcttatatc ccttgctctg gtcaaccagg ttgcagggtt tcctgtcctc acaggaacga
    10561 agtccctaaa gaaacagtgg cagccaggtt tagccccgga attgactgga ttcctttttt
    10621 agggcccatt ggtatggctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
    10681 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
    10741 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
    10801 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg
    10861 ggctgtccct gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga
    10921 acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa
    10981 tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat
    11041 tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga
    11101 cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac
    11161 gcagactatc tttctagggt taatctagct gcatcaggat catatcgtcg ggtctttttt
    11221 ccggctcagt catcgcccaa gctggcgcta tctgggcatc ggggaggaag aagcccgtgc
    11281 cttttcccgc gaggttgaag cggcatggaa agagtttgcc gaggatgact gctgctgcat
    11341 tgacgttgag cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc
    11401 ctttaacggt gaactgttcg ttcaggccac ctgggatacc agttcgtcgc ggcttttccg
    11461 gacacagttc cggatggtca gcccgaagcg catcagcaac ccgaacaata ccggcgacag
    11521 ccggaactgc cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg gatattacgt
    11581 cagcgaggac gggtatcctg gctggatgcc gcagaaatgg acatggatac cccgtgagtt
    11641 acccggcggg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
    11701 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
    11761 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
    11821 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
    11881 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
    11941 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
    12001 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
    12061 ttgctggcgt ttttccatag gctccgctcc cctgacgagc atcacaaaaa tcgacgctca
    12121 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
    12181 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
    12241 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
    12301 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
    12361 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
    12421 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
    12481 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
    12541 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
    12601 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
    12661 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
    12721 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
    12781 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
    12841 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
    12901 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
    12961 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
    13021 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
    13081 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
    13141 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
    13201 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
    13261 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
    13321 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
    13381 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
    13441 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
    13501 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
    13561 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
    13621 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
    13681 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
    13741 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
    13801 tttccccg
//
