LOCUS       Exported                7087 bp ds-DNA     circular SYN 11-SEP-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    PX854
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7087)
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7087
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..241
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        268..343
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /note="chicken beta-actin promoter"
     intron          1005..1233
                     /note="hybrid intron"
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1257..1277
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             1302..1622
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /note="FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1668..4052
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /note="Cas9(C)"
                     /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
                     IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
                     ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
                     LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
                     KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
                     AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
                     QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
                     FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             4053..4100
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    4134..4341
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4350..4490
                     /note="AAV2 ITR"
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4565..5020
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5302..5406
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             5407..6267
                     /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      6438..7026
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"