LOCUS       Exported                8430 bp DNA     circular SYN 08-MAY-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pX335
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8430)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8430
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        652..892
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        919..994
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1091..1376
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1378..1652
                     /label=chicken beta-actin promoter
     intron          1653..1880
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1901..1927
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1931..1951
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1961..6061
                     /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
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6065..6085
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6128..6335
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6344..6484
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6559..7014
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        7296..7400
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7401..8261
                     /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"
ORIGIN
        1 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
       61 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
      121 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
      181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
      241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
      301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
      361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tgagggccta
      661 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
      721 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
      781 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
      841 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
      901 gggtcttcga gaagacctgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
      961 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc tagaaatagc
     1021 aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca attctgcaga caaatggctc
     1081 tagaggtacc cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     1141 cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1201 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1261 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct
     1321 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     1381 aggtgagccc cacgttctgc ttcactctcc ccatctcccc ccctccccac ccccaatttt
     1441 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg
     1501 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
     1561 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
     1621 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
     1681 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
     1741 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
     1801 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
     1861 ctgaaatcac tttttttcag gttggaccgg tgccaccatg tacccatacg atgttccaga
     1921 ttacgcttcg ccgaagaaaa agcgcaaggt cgaagcgtcc gacaagaagt acagcatcgg
     1981 cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     2041 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     2101 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     2161 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     2221 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     2281 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     2341 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     2401 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     2461 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     2521 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     2581 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     2641 gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga gcctgggcct
     2701 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     2761 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     2821 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     2881 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     2941 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     3001 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     3061 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     3121 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     3181 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     3241 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     3301 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     3361 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     3421 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     3481 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     3541 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     3601 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     3661 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     3721 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     3781 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     3841 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     3901 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     3961 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     4021 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     4081 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     4141 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     4201 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     4261 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     4321 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     4381 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     4441 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     4501 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     4561 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     4621 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     4681 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     4741 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     4801 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     4861 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     4921 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     4981 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     5041 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     5101 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     5161 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     5221 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     5281 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     5341 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
     5401 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     5461 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     5521 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     5581 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
     5641 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     5701 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     5761 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     5821 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     5881 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     5941 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
     6001 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     6061 cagccccaag aagaagagaa aggtggaggc cagctaagaa ttcctagagc tcgctgatca
     6121 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     6181 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     6241 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     6301 gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg
     6361 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     6421 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     6481 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     6541 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     6601 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     6661 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     6721 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     6781 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     6841 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     6901 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     6961 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     7021 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     7081 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     7141 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     7201 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     7261 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     7321 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     7381 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     7441 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     7501 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     7561 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     7621 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     7681 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     7741 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     7801 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     7861 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     7921 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     7981 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     8041 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     8101 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     8161 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     8221 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     8281 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     8341 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     8401 cagaccccgt agaaaagatc aaaggatctt
//
