LOCUS Exported 8964 bp ds-DNA circular SYN 12-AUG-2018
DEFINITION synthetic circular DNA
KEYWORDS pX330A-1x2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8964)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8964
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 8..293
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 295..572
/note="chicken beta-actin promoter"
intron 573..801
/note="hybrid intron"
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 822..887
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 894..914
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 939..5039
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/note="Cas9"
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5040..5087
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
polyA_signal 5121..5328
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
repeat_region 5337..5477
/note="AAV2 ITR"
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5552..6007
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6289..6393
/gene="bla"
/note="AmpR promoter"
CDS 6394..7254
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 7425..8013
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 8075..8315
/note="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 8342..8417
/note="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
ORIGIN
1 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
421 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc
601 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
661 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg
721 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg
781 cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg
841 gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga
901 agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc
961 tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca
1021 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg
1081 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca
1141 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg
1201 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag
1261 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc
1321 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg
1381 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc
1441 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg
1501 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca
1561 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc
1621 tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga
1681 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg
1741 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc
1801 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga
1861 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc
1921 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag
1981 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc
2041 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac
2101 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca
2161 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt
2221 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc
2281 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga
2341 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc
2401 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc
2461 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat
2521 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg
2581 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact
2641 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg
2701 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca
2761 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca
2821 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga
2881 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg
2941 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg
3001 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc
3061 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg
3121 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga
3181 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga
3241 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca
3301 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg
3361 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg
3421 acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg
3481 acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca
3541 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg
3601 ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga
3661 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa
3721 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc
3781 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg
3841 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc
3901 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg
3961 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa
4021 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg
4081 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa
4141 ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca
4201 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt
4261 ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta
4321 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag
4381 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca
4441 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag
4501 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg
4561 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc
4621 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc
4681 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga
4741 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca
4801 aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata
4861 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca
4921 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc
4981 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca
5041 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag
5101 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
5161 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
5221 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
5281 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga
5341 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
5401 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
5461 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
5521 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
5581 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
5641 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
5701 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
5761 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
5821 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
5881 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
5941 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
6001 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
6061 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
6121 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
6181 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
6241 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
6301 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
6361 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
6421 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
6481 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
6541 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
6601 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
6661 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
6721 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
6781 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
6841 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
6901 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
6961 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
7021 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
7081 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
7141 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
7201 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
7261 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
7321 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
7381 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
7441 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
7501 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
7561 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
7621 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
7681 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
7741 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
7801 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
7861 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
7921 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
7981 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
8041 ttcctggcct tttgctggcc ttttgctcac atgtgagggc ctatttccca tgattccttc
8101 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
8161 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
8221 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
8281 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgggtctt cgagaagacc
8341 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
8401 tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta
8461 gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagagac cggcgccgct
8521 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
8581 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
8641 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta
8701 atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcc tccagctttt
8761 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
8821 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
8881 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
8941 ctttccaccg gtggtctctt ctag
//
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