LOCUS       sequence-412874-       16075 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ngn2 mediated cortical neuron induction, gateway destination, 
            Synapsin promoter BirA ligase expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16075)
  AUTHORS   Hallacli E, Kayatekin C, Nazeen S, Wang XH, Sheinkopf Z, Sathyakumar
            S, Sarkar S, Jiang X, Dong X, Di Maio R, Wang W, Keeney MT, Felsky 
            D, Sandoe J, Vahdatshoar A, Udeshi ND, Mani DR, Carr SA, Lindquist 
            S, De Jager PL, Bartel DP, Myers CL, Greenamyre JT, Feany MB, 
            Sunyaev SR, Chung CY, Khurana V
  TITLE     The Parkinson's disease protein alpha-synuclein is a modulator of 
            processing bodies and mRNA stability.
  JOURNAL   Cell. 2022 Jun 9;185(12):2035-2056.e33. doi: 
            10.1016/j.cell.2022.05.008.
  PUBMED    35688132
REFERENCE   2  (bases 1 to 16075)
  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"; doi: 
            "10.1016/j.cell.2022.05"; journalName: "Cell"; date: "2022-06-9- 9";
            volume: "185"; issue: "12"; pages: "2035-2056.e33"
COMMENT     Sequence Label: piggyBac_rtTA
            (4th_Gen)_Gateway_IRES_NGN2-2A-P...
FEATURES             Location/Qualifiers
     source          1..16075
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   132..166
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    719..943
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     932..949
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(972..1541)
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             complement(1560..1916)
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             complement(2346..2366)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2391..2540)
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             complement(3198..3251)
                     /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             complement(3261..3656)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(3666..4176)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3729..3747)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     protein_bind    4888..4906
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4924..4942
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4998..5016
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5044..5062
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5091..5109
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5127..5145
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5266..5390
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(5786..6091)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(5888..5907)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(6411..7094)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     7009..7028
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7095..7197)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(7375..7499)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    7551..8118
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    7568..8120
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7712..7729)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7939..7958
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             8132..8155
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             8948..9001
                     /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             9008..9607
                     /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(9008..9027)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9504..9524
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9608..10196
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9661..9681)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10079..10090)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    10211..10687
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(10410..10429)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     promoter        10735..11182
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             join(11196..11546,11547..11549,11550..12158)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             11196..12158
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     polyA_signal    12162..12210
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   13675..13737
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(13767..13787)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(13767..13785)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(13806..13822)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13806..13822)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13819..13841)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13830..13846
                     /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(13854..13860,13861..13878,13879..13884))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13899..13920
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14037..14054)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14208..14796)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14288..14307)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14967..15758,15759..15827))
                     /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     15590..15609
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(15828..15932)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(16007..16026)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
       61 gaatctgctt agggttaggc gttttgcgct gcttcgggga gatgggggag gctaactgag
      121 ctctagatta accctagaaa gatagtctgc gtaaaattga cgcatgcatt cttgaaatat
      181 tgctctctct ttctaaatag cgcgaatccg tcgctgtgca tttaggacat ctcagtcgcc
      241 gcttggagct cccgtgaggc gtgcttgtca atgcggtaag tgtcactgat tttgaactat
      301 aacgaccgcg tgagtcaaaa tgacgcatga ttatctttta cgtgactttt aagatttaac
      361 tcatacgata attatattgt tatttcatgt tctacttacg tgataactta ttatatatat
      421 attttcttgt tatagatatc acaaatgctt gtccttcctt cctgtaacac agccattaaa
      481 ccaggagcat cgcccttccc cggccctcag gtaagaggac caaataccgt agccgtttcc
      541 aatttcagtc ctttagcgcc acctggtgct aactactcta tcacgctttt atccaataac
      601 tacctttgta aatttccttt caaaagttct ggccgggcgc ggtggctcac gcttgtaatc
      661 ccagcacttt gtgaggggtc aggagttcga gacgatcaac tagaatgaat gtcgagaccc
      721 atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg
      781 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
      841 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
      901 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg
      961 gtttaaactc aaaacagaga tgtgtcgaag atggacagtc ctgtgctgat atgcatggca
     1021 tgcaagaggc actcgtcgtt caggaaggta tccagaatct cgttcaattc cggggtcagg
     1081 ggtgagtcca ggttcagatc ctcggtcatg gactcaagtg tggttgtcag ctcatccaga
     1141 tggccccttg ggggcggatg ggaaaggtcc atttggccac agattgcagc ctcttccttc
     1201 tggggaatca cagtatcggc catctcccga agggctttga cagcctggct cgtctcttca
     1261 tcgggatcct ccaacaggtg actggcctcg ggagtcactg cgggcgctgg atccagtggc
     1321 tgagggactg gtgccggggt cagtgacccg actggctcat gtactggacc ggttggtgtt
     1381 ggtgcgaggc tggcggggag tgggcggttg gcccatggac ttcctggagg atgaaatggc
     1441 cggattcgtt ttggctggca cacctcgcgg ccctcaaaca cgtcactaat agcggagccg
     1501 gcctcaggct tcggcaaaaa catcccttcc ctggaatccc ggctgccgct gccagagccc
     1561 agcaaggctg agaaatccat atcggcgata gagctgaagt cctcgtcgcc agacagcagt
     1621 ccattaggca ggcctggtgc tcccagaggg gcaggagctg gatcaggagg cctctgagcg
     1681 cctgtcacga gccgggtgat ggcctcgggg tattccatca gcatgggctc ggtggtgtga
     1741 ggggccacag ggatgccctg gttcagcagc tgctggaact cgctgttgtc cacgctggcc
     1801 aggtcggtga acacggcagg atcggtgctg tttcccagca gggctcccag atcctcgtcg
     1861 tcgaactgca gctgcagcag agcttcagac agtgtgccct cgccggcctg tgtaggttta
     1921 ggggctggtg gagccacagc ctgtggaggt ccaggagcca gcacaggcac gggtgctggt
     1981 gcctgagcca gtgcagacac catggctgga gctggtgcag gagcaggagc ctgaggcagc
     2041 acctgaggag gggctggagc cagagcagag gcctgagaga tctggccgct ggggaacacc
     2101 atggtaggga actcgtcgta gttgatggtg ctcaggctgc tggtgaaggg gtaaggctgg
     2161 ggggcaggtt ttggcacgct ggcgctggat ctgctgggca cggcgattct tctaggtgga
     2221 ggtctagggt cggtggggcc gctgaagggg gacttcttca tgatgctctt gaatgtctcg
     2281 taggtccgct tccgcttttc ctcgatccgg tgccggtcgt cggtgtcggg caggtactgg
     2341 ctaccaactt tgcgtttctt tttcggagat ccggaacttc tagagttaat cagcatgtcc
     2401 aggtcgaaat catcaagggc gtcactgccg agcatgtcga ggtcaaagtc atcaagggca
     2461 tccgaaccaa gcatgtcaag gtcaaaatca tcgagggcgt cacttcccag catatccaga
     2521 tcaaaatcgt ccaatgcgtc agcccgtccg gaaccgctgg cctccggccc gccgctttcg
     2581 cactttagct gtttctccag gccacatatg attagttcca ggccgaaaag gaaggcaggt
     2641 tcggctccct gccggtcgaa cagctcaatt gcttgtttca gaagtggggg catagaatcg
     2701 gtggtaggtg tctctctttc ctcttttgct acttgatgct cctgttcctc caatacgcag
     2761 cccagtgtaa agtggcccac ggcggacaga gcgtacagtg cgttctccag ggagaagcct
     2821 tgctgacaca ggaacgcgag ctgattttcc agggtttcgt actgtttctc tgttgggcgg
     2881 gtgccgagat gcactttagc cccgtcgcga tgtgagagga gagcacagcg gtatgacttg
     2941 gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca gggggcagga gtgggtatga
     3001 tgcctgtcca gcatctcgat tggcagggca tcgagcaggg cccgcttgtt cttcacgtgc
     3061 cagtacaggg taggctgctc aactcccagc ttttgagcga gtttccttgt cgtcaggcct
     3121 tcgataccga ctccattgag taattccaga gcagagttta tgactttgct cttgtccagt
     3181 ctagacatag aaccacctgg accagggttt tcttcaacat caccacaagt gaggagagaa
     3241 cctctacctt cgccggatcc gccctcccac acataaccag agggcagcaa ttcacgaatc
     3301 ccaactgccg tcggctgtcc atcactgtcc ttcactatgg ctttgatccc aggatgcaga
     3361 tcgagaagca cctgtcggca ccgtccgcag gggctcaaga tgcccctgtt ctcatttccg
     3421 atcgcgacga tacaagtcag gttgccagct gccgcagcag cagcagtgcc cagcaccacg
     3481 agttctgcac aaggtccccc agtaaaatga tatacattga caccagtgaa gatgcggccg
     3541 tcgctagaga gagctgcgct ggcgacgctg tagtcttcag agatggggat gctgttgatt
     3601 gtagccgttg ctctttcaat gagggtggat tcttcttgag acaaaggctt ggccatggtg
     3661 aattgctggg gagagaggtc ggtgattcgg tcaacgaggg agccgactgc cgacgtgcgc
     3721 tccggaggct tgcagaatgc ggaacaccgc gcgggcagga acagggccca cactaccgcc
     3781 ccacaccccg cctcccgcac cgccccttcc cggccgctgc tctcggcgcg ccctgctgag
     3841 cagccgctat tggccacagc ccatcgcggt cggcgcgctg ccattgctcc ctggcgctgt
     3901 ccgtctgcga gggtactagt gagacgtgcg gcttccgttt gtcacgtccg gcacgccgcg
     3961 aaccgcaagg aaccttcccg acttaggggc ggagcaggaa gcgtcgccgg ggggcccaca
     4021 agggtagcgg cgaagatccg ggtgacgctg cgaacggacg tgaagaatgt gcgagaccca
     4081 gggtcggcgc cgctgcgttt cccggaacca cgcccagagc agccgcgtcc ctgcgcaaac
     4141 ccagggctgc cttggaaaag gcgcaacccc aaccccgggg aggtggtccc tgcagttacg
     4201 ccaatgataa cccccgccag aaaaatctta gtagccttcc ctttttgttt tccgtgcccc
     4261 aactcggcgg attgactcgg ccccttccgg aaacacccga atcaacttct agtcaaatta
     4321 ttgttcacgc cgcaatgacc cacccctggc ccgcgtctgt ggaactgacc cctggtgtac
     4381 aggagagttc gctgctgaaa gtggtcccaa aggggtacta gtttttaagc tcccaactcc
     4441 ccctccccca gcgtctggag gattccacac cctcgcaccg caggggcgag gaagtgggcg
     4501 gagtccggtt ttggcgccag ccgctgaggc tgccaagcag aaaagccacc gctgaggaga
     4561 ctccggtcac tgtcctcgcc ccgcctcccc cttccctccc cttggggacc accgggcgcc
     4621 acgccgcgaa cggtaagtgc cgcggtcgtc ggcgcctccg ccctccccct agggccccaa
     4681 ttcccagcgg gcgcggcgcg cggcccctcc ccccgccggg cgcgcgcccg ctgccccgcc
     4741 cttcgtggcc gcccggcgtg ggcggtgcca cccctccccc cggcggcccc gcgcgcagct
     4801 cccggctccc tcccccttcg gatgtggctt gagctgtagg cgcggaggct agctggggat
     4861 ctggtgggct ctattggtag actttagtcc ctatcagtga tagagaaggt atctccagtt
     4921 tactccctat cagtgataga gacatccatg gaatcaactt cagctttatg agtaagaagc
     4981 cctataggat gtggctttcc ctatcagtga tagagaacta atgataagac cactgcactt
     5041 tagtccctat cagtgataga gatgagatct tctggtgtac aggagttttc tccctatcag
     5101 tgatagagag tctgtgtgcc atttgatccc tatcagtgat agagactgta tttccaggta
     5161 ggcctgtaca gtgggaggcc tatataagca gagctccttt agtgaaccct cagatggcca
     5221 gagataagag agaaaagaag agtaagaaga aatataagag ccaccacaag tttgtacaaa
     5281 aaagctgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     5341 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gtcgaccgac
     5401 agccttccaa atgttcttcg ggtgatgctg ccaacttagt cgaccgacag ccttccaaat
     5461 gttcttctca aacggaatcg tcgtatccag cctactcgct attgtcctca atgccgtatt
     5521 aaatcataaa aagaaataag aaaaagaggt gcgagcctct tttttgtgtg acaaaataaa
     5581 aacatctacc tattcatata cgctagtgtc atagtcctga aaatcatctg catcaagaac
     5641 aatttcacaa ctcttatact tttctcttac aagtcgttcg gcttcatctg gattttcagc
     5701 ctctatactt actaaacgtg ataaagtttc tgtaatttct actgtatcga cctgcagact
     5761 ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt
     5821 tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gagaccggca
     5881 cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt
     5941 aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc
     6001 gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct
     6061 ctcttttata ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa
     6121 gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc
     6181 cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag
     6241 atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa
     6301 tacgctgctt catagcatac ctctttttga catacttcgg gtatacatat cagtatatat
     6361 tcttataccg caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc
     6421 ggatccacgc ggcgtttacg ccccccctgc cactcatcgc agtactgttg taattcatta
     6481 agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc
     6541 atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag
     6601 ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag
     6661 acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac
     6721 gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag
     6781 agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc
     6841 catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg
     6901 cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt
     6961 aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga
     7021 aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg
     7081 atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg
     7141 cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg
     7201 tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt
     7261 atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt
     7321 gatgctgcca acttagtcga ctacaggtca ctaataccat ctaagtagtt gattcatagt
     7381 gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt
     7441 aatatattga tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggtt
     7501 gatctgctgg aagatggcga ttagagaggt acgaagatgg cgattagaga tccctccccc
     7561 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     7621 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     7681 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     7741 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     7801 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     7861 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     7921 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     7981 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     8041 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     8101 tgaaaaacac gatgataata tggccacaat ggactacaag gacgacgatg acaagttcgt
     8161 caaatctgag actctggagt tgaaggagga agaggaggta ctgatgctgc tgggctcggc
     8221 ttccccggcc tcggcgaccc tgaccccgat gtcctccagc gcggacgagg aggaggacga
     8281 ggagctgcgc cggccgggct ccgcgcgtgg gcagcgtgga gcggaagccg ggcagggggt
     8341 gcagggcagt ccggcgtcgg gtgccggggg ttgccggcca gggcggctgc tgggcctgat
     8401 gcacgagtgc aagcgtcgcc cgtcgcgctc acgggccgtc tcccgaggtg ccaagacggc
     8461 ggagacggtg cagcgcatca agaagacccg caggctcaag gccaacaacc gcgagcgcaa
     8521 ccgcatgcac aacctaaacg ccgcgctgga cgcgctgcgc gaggtgctgc ccaccttccc
     8581 cgaggatgcc aagctcacga agatcgagac gctgcgcttc gcccacaatt acatctgggc
     8641 gctcaccgag actctgcgcc tggcggacca ctgcgccggc gccggtggcc tccagggggc
     8701 gctcttcacg gaggcggtgc tcctgagccc gggagctgcg ctcggcgcca gcggggacag
     8761 cccttctcca ccttcctcct ggagctgcac caacagcccg gcgtcatcct ccaactccac
     8821 gtccccatac agctgcactt tatcgcccgc tagccccggg tcagacgtgg actactggca
     8881 gcccccacct ccggagaagc atcgttatgc gcctcacctg cccctcgcca gggactgtat
     8941 ctctagagag ggcaggggaa gtcttctaac atgcggggac gtggaggaaa atcccggccc
     9001 actcgagatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     9061 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     9121 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     9181 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     9241 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     9301 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     9361 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     9421 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     9481 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     9541 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     9601 tgcctgaaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     9661 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     9721 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     9781 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     9841 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     9901 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     9961 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
    10021 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
    10081 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
    10141 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctcg
    10201 agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
    10261 tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
    10321 ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
    10381 tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc
    10441 acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc
    10501 tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
    10561 gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat
    10621 ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc
    10681 tgtccttgta ccttaattaa gttaactgct agactgcaga gggccctgcg tatgagtgca
    10741 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
    10801 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
    10861 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
    10921 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
    10981 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
    11041 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
    11101 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
    11161 cgtgtcgtgc ctgagagcgc agtcgagccg ccaccatgaa ggacaacacc gtgcccctga
    11221 agctgatcgc cctgctggcc aacggcgagt tccactctgg cgagcagctg ggagagaccc
    11281 tgggaatgag cagagccgcc atcaacaagc acatccagac actgagagac tggggagtgg
    11341 acgtgttcac cgtgcctggc aagggctaca gcctgcctga gcctatccag ctgctgaacg
    11401 ccaagcagat cctgggacag ctggatggcg gaagcgtggc cgtgctgcct gtgatcgact
    11461 ccaccaatca gtacctgctg gacagaatcg gagagctgaa gtccggcgac gcctgcatcg
    11521 ccgagtacca gcaggctggc agaggaagaa gaggacggaa gtggttcagc ccattcggag
    11581 ccaacctgta cctgtccatg ttctggagac tggagcaggg acctgctgct gccatcggac
    11641 tgagtctggt gatcggaatc gtgatggccg aggtgctgag aaagctggga gccgacaagg
    11701 tgagagtgaa gtggcctaat gacctgtacc tccaggaccg caagctggct ggcatcctgg
    11761 tggagctgac aggcaagaca ggcgatgccg ctcagatcgt gatcggagcc ggaatcaaca
    11821 tggccatgag aagagtggag gagagcgtgg tgaaccaggg ctggatcacc ctgcaggagg
    11881 ctggcatcaa cctggaccgg aacaccctgg ccgccatgct gatcagagag ctgagagccg
    11941 ctctggagct gttcgagcag gagggactgg ctccttacct gagcagatgg gagaagctgg
    12001 acaacttcat caacagacct gtgaagctga tcatcggcga caaggaaatc ttcggcatct
    12061 ccagaggaat cgacaagcag ggagctctgc tgctggagca ggacggaatc atcaagccct
    12121 ggatgggcgg agaaatctcc ctgagaagcg cagagaagta acggcaataa aaagacagaa
    12181 taaaacgcac gggtgttggg tcgtttgttc gcggccgcga gctcacgggg acagcccccc
    12241 ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag cagcgagccg
    12301 cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca gcgtgcgggg
    12361 acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct tctcgctgct
    12421 ctttgagcct gcagacacct ggggggatac ggggaaaaag ctttaggctg aaagagagat
    12481 ttagaatgac agaatcatag aacggcctgg gttgcaaagg agcacagtgc tcatccagat
    12541 ccaaccccct gctatgtgca gggtcatcaa ccagcagccc aggctgccca gagccacatc
    12601 cagcctggcc ttgaatgcct gcagggatgg ggcatccaca gcctccttgg gcaacctgtt
    12661 cagtgcgtca ccaccctctg ggggaaaaac tgcctcctca tatccaaccc aaacctcccc
    12721 tgtctcagtg taaagccatt cccccttgtc ctatcaaggg ggagtttgct gtgacattgt
    12781 tggtctgggg tgacacatgt ttgccaattc agtgcatcac ggagaggcag atcttgggga
    12841 taaggaagtg caggacagca tggacgtggg acatgcaggt gttgagggct ctgggacact
    12901 ctccaagtca cagcgttcag aacagcctta aggataagaa gataggatag aaggacaaag
    12961 agcaagttaa aacccagcat ggagaggagc acaaaaaggc cacagacact gctggtccct
    13021 gtgtctgagc ctgcatgttt gatggtgtct ggatgcaagc agaaggggtg gaagagcttg
    13081 cctggagaga tacagctggg tcagtaggac tgggacaggc agctggagaa ttgccatgta
    13141 gatgttcata caatcgtcaa atcatgaagg ctggaaaagc cctccaagat ccccaagacc
    13201 aaccccaacc cacccaccgt gcccactggc catgtccctc agtgccacat ccccacagtt
    13261 cttcatcacc tccagggacg gtgacccccc cacctccgtg ggcagctgtg ccactgcagc
    13321 accgctcttt ggagaaggta aatcttgcta aatccagccc gaccctcccc tggcacaacg
    13381 taaggccatt atctctcatc caactccagg acggagtcag tgagaatatt gtttaaacat
    13441 tataggcgcg ccattgctgg gattacaggc gtgaaccact gctcccttcc ctgtcctttt
    13501 aaaagttttg ttactttata gaagaaattt tgagtttttg ttttttttta ataaataaat
    13561 aaacataaat aaattgtttg ttgaatttat tattagtatg taagtgtaaa tataataaaa
    13621 cttaatatct attcaaatta ataaataaac ctcgatatac agaccgataa aacacatgcg
    13681 tcaattttac gcatgattat ctttaacgta cgtcacaata tgattatctt tctagggtta
    13741 agcgatcgct aatccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
    13801 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
    13861 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
    13921 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
    13981 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
    14041 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
    14101 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
    14161 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
    14221 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
    14281 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
    14341 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
    14401 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
    14461 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
    14521 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
    14581 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
    14641 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
    14701 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
    14761 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
    14821 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
    14881 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
    14941 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
    15001 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
    15061 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
    15121 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
    15181 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
    15241 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
    15301 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
    15361 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
    15421 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
    15481 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
    15541 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
    15601 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
    15661 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
    15721 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
    15781 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
    15841 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
    15901 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
    15961 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
    16021 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctga
//
