LOCUS       sequence-179981-       10469 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression plasmid for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10469)
  AUTHORS   Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, 
            DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, 
            Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F
  TITLE     Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.
  JOURNAL   Nat Biotechnol. 2017 Jan;35(1):31-34. doi: 10.1038/nbt.3737. Epub 
            2016 Dec 5.
  PUBMED    27918548
REFERENCE   2  (bases 1 to 10469)
  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"; doi: "10.1038/nbt.3737"; 
            journalName: "Nat Biotechnol"; date: "2017-01"; volume: "35"; issue:
            "1"; pages: "31-34"
COMMENT     Sequence Label: 6His-MBP-TEV-huLbCpf1
FEATURES             Location/Qualifiers
     source          1..10469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     939..958
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(978..1055)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1261..1280
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1364..1383
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1364..1382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1383..1407
                     /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)."
     RBS             1422..1444
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1458..1475
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2430..2439
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2592..2609
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2616..2636
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2637..6320
                     /codon_start=1
                     /product="CRISPR-associated protein from Lachnospiraceae 
                     bacterium ND2006 (Zetsche et al., 2015)"
                     /label=LbCpf1
                     /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE
                     DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE
                     IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFS
                     EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG
                     EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL
                     SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN
                     GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ
                     LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD
                     LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS
                     KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG
                     NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL
                     IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV
                     EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK
                     EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI
                     NTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD
                     YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG
                     FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ
                     NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD
                     YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ
                     QGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRN
                     YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS
                     VKH"
     CDS             6321..6368
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6375..6455
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             6480..6503
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6504..6548
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6564..6581
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6634..6652)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6648..6695
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6732..7187
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6819..6838)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7029..7050
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7213..7317
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7318..7386,7387..8178)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7536..7555)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8349..8937
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8838..8857
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9091..9108
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    9123..9262
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9188..9207)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9348..9370
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(9364..9555)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(10364..10469,1..977))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
       61 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
      121 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
      181 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
      241 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
      301 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
      361 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
      421 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
      481 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
      541 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
      601 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
      661 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
      721 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
      781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
      841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
      901 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
      961 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     1021 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     1081 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     1141 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     1201 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     1261 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     1321 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     1381 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     1441 gagatataca tatgggtcat catcatcacc atcacaaaat cgaagaaggc aaactggtta
     1501 tttggatcaa tggcgataaa ggctataatg gtctggcaga agttggcaaa aaattcgaaa
     1561 aagataccgg cattaaagtg accgttgaac atccggataa actggaagaa aaatttccgc
     1621 aggttgcagc aaccggtgat ggtccggata ttatcttttg ggcacatgat cgttttggtg
     1681 gttatgcaca gagcggtctg ctggcagaaa ttacaccgga taaagcattt caggacaaac
     1741 tgtatccgtt tacctgggat gcagttcgct ataacggtaa actgattgca tatccgattg
     1801 cagttgaagc actgagcctg atctataaca aagatctgct gccgaatccg cctaaaacct
     1861 gggaagaaat tccggcactg gataaagaac tgaaagcaaa aggtaaaagc gcactgatgt
     1921 ttaatctgca agaaccgtat tttacctggc ctctgattgc agcagatggt ggctatgcat
     1981 tcaaatatga aaacggcaaa tatgatatca aagacgtggg tgttgataat gcgggtgcaa
     2041 aagccggtct gacctttctg gttgatctga ttaaaaacaa acacatgaac gccgataccg
     2101 attatagcat tgcagaagca gcatttaaca aaggtgaaac cgcaatgaca attaatggtc
     2161 cgtgggcatg gtcaaatatt gataccagca aagtgaatta tggtgttacc gttctgccga
     2221 catttaaagg tcagccgagc aaaccgtttg ttggtgttct gagcgcaggt attaatgcag
     2281 caagcccgaa caaagaactg gcaaaagaat ttctggaaaa ctatctgctg accgatgaag
     2341 gtctggaagc agtgaataaa gataaaccgc tgggtgcagt tgcactgaaa agctatgaag
     2401 aagaactggt taaagatccg cgtattgcag ccaccatgga aaatgcacag aaaggcgaaa
     2461 ttatgccgaa tattccgcag atgagcgcat tttggtatgc cgttcgtacc gcagtgatta
     2521 atgccgcatc aggtcgtcag accgttgatg aagccctgaa agatgcccag accggtgcaa
     2581 gcaccggatc cctggttccg cgtggtagcg ctagcgaaaa tctgtatttt cagggaatga
     2641 gcaagctgga gaagtttaca aactgctact ccctgtctaa gaccctgagg ttcaaggcca
     2701 tccctgtggg caagacccag gagaacatcg acaataagcg gctgctggtg gaggacgaga
     2761 agagagccga ggattataag ggcgtgaaga agctgctgga tcgctactat ctgtctttta
     2821 tcaacgacgt gctgcacagc atcaagctga agaatctgaa caattacatc agcctgttcc
     2881 ggaagaaaac cagaaccgag aaggagaata aggagctgga gaacctggag atcaatctgc
     2941 ggaaggagat cgccaaggcc ttcaagggca acgagggcta caagtccctg tttaagaagg
     3001 atatcatcga gacaatcctg ccagagttcc tggacgataa ggacgagatc gccctggtga
     3061 acagcttcaa tggctttacc acagccttca ccggcttctt tgataacaga gagaatatgt
     3121 tttccgagga ggccaagagc acatccatcg ccttcaggtg tatcaacgag aatctgaccc
     3181 gctacatctc taatatggac atcttcgaga aggtggacgc catctttgat aagcacgagg
     3241 tgcaggagat caaggagaag atcctgaaca gcgactatga tgtggaggat ttctttgagg
     3301 gcgagttctt taactttgtg ctgacacagg agggcatcga cgtgtataac gccatcatcg
     3361 gcggcttcgt gaccgagagc ggcgagaaga tcaagggcct gaacgagtac atcaacctgt
     3421 ataatcagaa aaccaagcag aagctgccta agtttaagcc actgtataag caggtgctga
     3481 gcgatcggga gtctctgagc ttctacggcg agggctatac atccgatgag gaggtgctgg
     3541 aggtgtttag aaacaccctg aacaagaaca gcgagatctt cagctccatc aagaagctgg
     3601 agaagctgtt caagaatttt gacgagtact ctagcgccgg catctttgtg aagaacggcc
     3661 ccgccatcag cacaatctcc aaggatatct tcggcgagtg gaacgtgatc cgggacaagt
     3721 ggaatgccga gtatgacgat atccacctga agaagaaggc cgtggtgacc gagaagtacg
     3781 aggacgatcg gagaaagtcc ttcaagaaga tcggctcctt ttctctggag cagctgcagg
     3841 agtacgccga cgccgatctg tctgtggtgg agaagctgaa ggagatcatc atccagaagg
     3901 tggatgagat ctacaaggtg tatggctcct ctgagaagct gttcgacgcc gattttgtgc
     3961 tggagaagag cctgaagaag aacgacgccg tggtggccat catgaaggac ctgctggatt
     4021 ctgtgaagag cttcgagaat tacatcaagg ccttctttgg cgagggcaag gagacaaaca
     4081 gggacgagtc cttctatggc gattttgtgc tggcctacga catcctgctg aaggtggacc
     4141 acatctacga tgccatccgc aattatgtga cccagaagcc ctactctaag gataagttca
     4201 agctgtattt tcagaaccct cagttcatgg gcggctggga caaggataag gagacagact
     4261 atcgggccac catcctgaga tacggctcca agtactatct ggccatcatg gataagaagt
     4321 acgccaagtg cctgcagaag atcgacaagg acgatgtgaa cggcaattac gagaagatca
     4381 actataagct gctgcccggc cctaataaga tgctgccaaa ggtgttcttt tctaagaagt
     4441 ggatggccta ctataacccc agcgaggaca tccagaagat ctacaagaat ggcacattca
     4501 agaagggcga tatgtttaac ctgaatgact gtcacaagct gatcgacttc tttaaggata
     4561 gcatctcccg gtatccaaag tggtccaatg cctacgattt caacttttct gagacagaga
     4621 agtataagga catcgccggc ttttacagag aggtggagga gcagggctat aaggtgagct
     4681 tcgagtctgc cagcaagaag gaggtggata agctggtgga ggagggcaag ctgtatatgt
     4741 tccagatcta taacaaggac ttttccgata agtctcacgg cacacccaat ctgcacacca
     4801 tgtacttcaa gctgctgttt gacgagaaca atcacggaca gatcaggctg agcggaggag
     4861 cagagctgtt catgaggcgc gcctccctga agaaggagga gctggtggtg cacccagcca
     4921 actcccctat cgccaacaag aatccagata atcccaagaa aaccacaacc ctgtcctacg
     4981 acgtgtataa ggataagagg ttttctgagg accagtacga gctgcacatc ccaatcgcca
     5041 tcaataagtg ccccaagaac atcttcaaga tcaatacaga ggtgcgcgtg ctgctgaagc
     5101 acgacgataa cccctatgtg atcggcatcg ataggggcga gcgcaatctg ctgtatatcg
     5161 tggtggtgga cggcaagggc aacatcgtgg agcagtattc cctgaacgag atcatcaaca
     5221 acttcaacgg catcaggatc aagacagatt accactctct gctggacaag aaggagaagg
     5281 agaggttcga ggcccgccag aactggacct ccatcgagaa tatcaaggag ctgaaggccg
     5341 gctatatctc tcaggtggtg cacaagatct gcgagctggt ggagaagtac gatgccgtga
     5401 tcgccctgga ggacctgaac tctggcttta agaatagccg cgtgaaggtg gagaagcagg
     5461 tgtatcagaa gttcgagaag atgctgatcg ataagctgaa ctacatggtg gacaagaagt
     5521 ctaatccttg tgcaacaggc ggcgccctga agggctatca gatcaccaat aagttcgaga
     5581 gctttaagtc catgtctacc cagaacggct tcatctttta catccctgcc tggctgacat
     5641 ccaagatcga tccatctacc ggctttgtga acctgctgaa aaccaagtat accagcatcg
     5701 ccgattccaa gaagttcatc agctcctttg acaggatcat gtacgtgccc gaggaggatc
     5761 tgttcgagtt tgccctggac tataagaact tctctcgcac agacgccgat tacatcaaga
     5821 agtggaagct gtactcctac ggcaaccgga tcagaatctt ccggaatcct aagaagaaca
     5881 acgtgttcga ctgggaggag gtgtgcctga ccagcgccta taaggagctg ttcaacaagt
     5941 acggcatcaa ttatcagcag ggcgatatca gagccctgct gtgcgagcag tccgacaagg
     6001 ccttctactc tagctttatg gccctgatga gcctgatgct gcagatgcgg aacagcatca
     6061 caggccgcac cgacgtggat tttctgatca gccctgtgaa gaactccgac ggcatcttct
     6121 acgatagccg gaactatgag gcccaggaga atgccatcct gccaaagaac gccgacgcca
     6181 atggcgccta taacatcgcc agaaaggtgc tgtgggccat cggccagttc aagaaggccg
     6241 aggacgagaa gctggataag gtgaagatcg ccatctctaa caaggagtgg ctggagtacg
     6301 cccagaccag cgtgaagcac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
     6361 agaaaaaggg atcctaccca tacgatgttc cagattacgc ttatccctac gacgtgcctg
     6421 attatgcata cccatatgat gtccccgact atgcctaaag cctcgagggt ggtggtagcg
     6481 attataaaga tgatgatgat aaaggcctga acgatatctt tgaagcccag aaaattgaat
     6541 ggcacgagta aaagcttgtc gagcaccacc accaccacca ctgagatccg gctgctaaca
     6601 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     6661 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     6721 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     6781 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     6841 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     6901 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     6961 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     7021 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     7081 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     7141 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     7201 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     7261 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     7321 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     7381 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     7441 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     7501 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     7561 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     7621 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     7681 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     7741 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     7801 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     7861 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     7921 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     7981 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggtagtcgc
     8041 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     8101 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     8161 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     8221 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     8281 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     8341 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     8401 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     8461 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     8521 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     8581 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     8641 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     8701 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     8761 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     8821 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     8881 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     8941 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     9001 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     9061 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     9121 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     9181 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     9241 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     9301 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     9361 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     9421 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     9481 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     9541 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     9601 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     9661 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     9721 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     9781 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     9841 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     9901 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     9961 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
    10021 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
    10081 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
    10141 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
    10201 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
    10261 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
    10321 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
    10381 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
    10441 tttgcgtatt gggcgccagg gtggttttt
//
