A621-Cbh-367Cterm-U6-gRNA

LOCUS       A621-Cbh-367Cterm-U6-gRNA          7661 bp DNA     circular SYN 02-APR-2026
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7661)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..7661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             18..74
                     /codon_start=1
                     /standard_name="SV40 NLS"
                     /product="nuclear localization signal of SV40 (simianvirus 
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="MKRTADGSEFESPKKKRKV"
     CDS             75..227
                     /codon_start=1
                     /label=RmaDnaBIntC
                     /translation="AAACPELRQLAQSDVYWDPIVSIEPDGVEEVFDLTVPGPHNFVAN
                     DIIAHN"
     CDS             228..3230
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N368-1368)
                     /translation="SQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH
                     QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE
                     TITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYV
                     TEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA
                     SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM
                     KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED
                     IQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE
                     NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD
                     QELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR
                     QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY
                     DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP
                     KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
                     LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI
                     ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
                     DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYL
                     ASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
                     RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY
                     ETRIDLSQLGGD"
     primer_bind     491..514
                     /label=68-Cas9-F2
     primer_bind     complement(700..719)
                     /label=114-WKG-338-R1
     primer_bind     1332..1353
                     /label=69-Cas9-F3
     primer_bind     2138..2158
                     /label=70-Cas9-F4
     primer_bind     2702..2721
                     /label=71-Cas9-F5
     primer_bind     2972..2988
                     /label=482-Cas9-F
     CDS             3243..3293
                     /codon_start=1
                     /product="Better than SV40"
                     /label=BP-SV40 NLS
                     /translation="KRTADGSEFEPKKKRKV"
     misc_feature    3300..3396
                     /label=SV40 polyA
     ncRNA           complement(3409..3484)
                     /standard_name="RNA scaffold"
                     /label=RNA scaffold
     regulatory      complement(3505..3768)
                     /standard_name="hU6 promoter"
                     /label=hU6 promoter
                     /note="Geneious type: promoter"
     primer_bind     complement(3563..3582)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3733..3753)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   3777..3906
                     /label=AAV2 ITR
     primer_bind     complement(3823..3834)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3884..3895
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3925..3942)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3930..3948)
                     /label=43-WKG254-F
     primer_bind     complement(3934..3956)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4083..4538
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4097..4121)
                     /label=184-WKG-599-R
     primer_bind     complement(4140..4159)
                     /label=121-WKG-450-F1
     primer_bind     complement(4170..4189)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4172..4192)
                     /label=122-WKG-465-F
     primer_bind     4380..4401
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4382..4401
                     /label=189-WKG-612-F
     CDS             4669..5529
                     /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     complement(4887..4906)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(5161..5183)
                     /label=198-WKG-623-F4
     rep_origin      5700..6288
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5776..5804)
                     /label=197-WKG-623-F3
     primer_bind     6177..6201
                     /label=225-WKG-1079-F
     primer_bind     6189..6208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6303..6322
                     /label=422-PM1195-F1
     primer_bind     6367..6393
                     /label=79-WKG-304-F1
     primer_bind     complement(6395..6414)
                     /label=196-WKG-623-F2
     primer_bind     6442..6459
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6508..6542
                     /label=139-WKG-445-R12
     protein_bind    6576..6597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     6599..6621
                     /label=154-lacUV5 prom-For
     primer_bind     6606..6629
                     /label=140-WKG-448-R1
     primer_bind     6606..6629
                     /label=190-WKG-612-R
     primer_bind     6609..6631
                     /label=41-lac-R
     promoter        6612..6642
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6650..6666
                     /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)."
     primer_bind     6655..6677
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6674..6690
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6679..6694
                     /label=152-M13-Rev
     repeat_region   6717..6846
                     /label=AAV2 ITR
     primer_bind     complement(6728..6739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6789..6800
                     /label=L4440
                     /note="L4440 vector, forward primer"
     regulatory      join(6863..7661,1..5)
                     /standard_name="CBh promoter"
                     /label=CBh promoter
                     /note="Geneious type: promoter"
     primer_bind     7117..7155
                     /label=217-WKG-278-F
     primer_bind     7321..7341
                     /label=1-CAG-F
ORIGIN
        1 gttggaccgg tgccaccatg aaacggacag ccgacggaag cgagttcgag tcaccaaaga
       61 agaagcggaa agtcgcggcg gcgtgcccgg aactgcgtca gctggcgcag agcgatgtgt
      121 attgggatcc gattgtgagc attgaaccgg atggcgtgga agaagtgttt gatctgaccg
      181 tgccgggccc gcataacttt gtggcgaacg atattattgc gcataacagc caggaagagt
      241 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
      301 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
      361 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
      421 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
      481 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
      541 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
      601 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
      661 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
      721 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
      781 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
      841 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
      901 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
      961 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     1021 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     1081 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     1141 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     1201 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     1261 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     1321 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     1381 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     1441 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     1501 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     1561 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     1621 ggctgtccga ctacgatgtg gacgctatcg tgcctcagag ctttctgaag gacgactcca
     1681 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     1741 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     1801 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     1861 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     1921 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     1981 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     2041 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     2101 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     2161 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     2221 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     2281 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     2341 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     2401 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     2461 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     2521 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     2581 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     2641 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     2701 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     2761 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     2821 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     2881 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     2941 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     3001 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     3061 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     3121 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     3181 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggtgac tctggcggct
     3241 caaaaagaac cgccgacggc agcgaattcg agcccaagaa gaagaggaaa gtctaagatc
     3301 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     3361 caagttaaca acaacaattg cattcatttt atgttttcga gaaaaaaagc accgactcgg
     3421 tgccactttt tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta
     3481 aaacacagaa ccagacagcc atgcggtgtt tcgtcctttc cacaagatat ataaagccaa
     3541 gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa aacataattt
     3601 taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat
     3661 ctttgtgttt acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg
     3721 caaatatgaa ggaatcatgg gaaataggcc ctcttcctgc ccgaccttgc ggccgcagga
     3781 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     3841 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     3901 gcgcagcctt aattaaccta attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     3961 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     4021 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4081 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4141 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4201 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4261 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     4321 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4381 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4441 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4501 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     4561 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4621 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4681 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4741 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4801 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4861 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4921 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4981 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5041 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5101 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5161 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5221 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5281 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5341 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5401 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5461 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5521 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5581 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5641 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5701 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5761 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5821 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5881 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5941 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6001 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6061 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6121 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6181 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6241 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6301 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6361 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6421 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     6481 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     6541 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     6601 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     6661 taacaatttc acacaggaaa cagctatgac catgattacg ccagatttaa ttaaggctgc
     6721 gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg acctttggtc
     6781 gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
     6841 gttcctgcgg cctctagatc agggtacccg ttacataact tacggtaaat ggcccgcctg
     6901 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc
     6961 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     7021 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     7081 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     7141 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
     7201 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
     7261 cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
     7321 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     7381 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
     7441 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     7501 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     7561 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
     7621 ttaattacct ggagcacctg cctgaaatca ctttttttca g
//