A665 CMV-1115Cterm-RT-U6-gRNA-U6-pegRNA

LOCUS       A665 CMV-1115Cterm-RT-U6-gRNA-U6-pegRNA          7605 bp DNA     circular SYN 01-APR-2026
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7605)
  AUTHORS   Hemiao
  TITLE     Direct Submission
  JOURNAL   Exported Aug 31, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..7605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(27..43)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    51..67
                     /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(75..105)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    120..141
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(429..1017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1188..2048)
                     /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"
     promoter        complement(2049..2153)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2179..2634)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     2776..2792
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2964..3212
                     /label=U6 promoter
     misc_feature    3358..3394
                     /label=evopreq1-1 trimmed
     promoter        3402..3715
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     misc_feature    3737..3822
                     /label=gRNA-Opt
     polyA_signal    3836..4060
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(4321..4371)
                     /codon_start=1
                     /product="Better than SV40"
                     /label=BP-SV40(BE4Max2)
                     /translation="KRTADGSEFEPKKKRKV"
     CDS             complement(4384..5871)
                     /codon_start=1
                     /label=MMLVRanseH-MMLV(D200N,L603W,T330P,T306K,W313F,H8Y)
                     /translation="TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQ
                     APLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKP
                     GTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT
                     SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQY
                     VDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT
                     EARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQ
                     QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL
                     DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMT
                     HYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCL"
     CDS             complement(5899..5946)
                     /codon_start=1
                     /label=XTEN(16aa,DE4Max,DavidLiu)
                     /translation="SGSETPGTSESATPES"
     CDS             complement(5971..6729)
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9-C(1116-1368)
                     /translation="SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV
                     KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE
                     LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK
                     RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT
                     STKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(6730..6882)
                     /codon_start=1
                     /label=RmaDnaBIntC
                     /translation="AAACPELRQLAQSDVYWDPIVSIEPDGVEEVFDLTVPGPHNFVAN
                     DIIAHN"
     CDS             complement(6883..6936)
                     /codon_start=1
                     /label=NLS(BE4Max1)
                     /translation="KRTADGSEFESPKKKRKV"
     promoter        complement(6964..7167)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        7168..7453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ccttaattaa atctggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
       61 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
      121 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
      181 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
      241 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
      301 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
      361 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
      421 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
      481 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
      541 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
      601 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
      661 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
      721 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
      781 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
      841 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
      901 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
      961 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     1021 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     1081 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     1141 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     1201 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     1261 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     1321 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     1381 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     1441 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     1501 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     1561 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     1621 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     1681 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     1741 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     1801 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     1861 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     1921 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     1981 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     2041 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     2101 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     2161 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     2221 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     2281 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     2341 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2401 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2461 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2521 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2581 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     2641 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     2701 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     2761 cccagtcacg acgttgtaaa acgacggcca gtgaattagg ttaattaagg ctgcgcgctc
     2821 gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg
     2881 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
     2941 gcggccgcaa ggtcgggcag gaagagggcc tatttcccat gattccttca tatttgcata
     3001 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
     3061 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     3121 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     3181 ctttatatat cttgtggaaa ggacgaaaca ccggagatct ctgaagtgaa gagtttaaga
     3241 gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa
     3301 gtgggaccga gtcggtccgg aattctgtat ccatcttcac ttcagagata ataagaccgc
     3361 ggttctatct agttacgcgt taaaccaact agaatttttt tgatccgacg ccgccatctc
     3421 taggcccgcg ccggccccct cgcacagact tgtgggagaa gctcggctac tcccctgccc
     3481 cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac
     3541 agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa
     3601 gagatacaaa tactaaatta ttattttaaa aaacagcaca aaaggaaact caccctaact
     3661 gtaaagtaat tgtgtgtttt gagactataa atatcccttg gagaaaagcc ttgttgagac
     3721 aaacagtagt ggaaaggttt aagagctatg ctggaaacag catagcaagt ttaaataagg
     3781 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc tcgagccata
     3841 gagcccaccg catccccagc atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc
     3901 tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc
     3961 tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg
     4021 gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcgcaccacg gaattgtcag
     4081 tgcccaacag ccgagcccct gtccagcagc gggcaaggca ggcggcgatg agttccgccg
     4141 tggcaagaac taaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa
     4201 tagcatgata caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt
     4261 taagaatacc agtcaatctt tcacaaattt tgtaatccag aggttgatta tcagatctta
     4321 gactttcctc ttcttcttgg gctcgaattc gctgccgtcg gcggttcttt ttgagccgcc
     4381 agaaaggcag ttgtgttgca gcccttcctc aggcagtggg agcagcgtag ccgggttcag
     4441 ggctaccacc ggtccgaact ggacccggtc cgtgtccaaa agcaaggcct gatagtgagt
     4501 catccgggcg ttggaaagcc agcggtcggg gggttgtttg actagtgcct ctactgcatg
     4561 gggggccaga atgactagtg gctgtcccat ggttagcttg cctgcatcct ttgtcagtac
     4621 ggcaatggct gctaccatcc gtaggcaagg gggccaccca gctgctactg ggtctagctt
     4681 tttggacagg taggccaccg gccgacgcca aggtcccagt ttttgcgtta ggacaccttt
     4741 ggcgtagccc tgcttctcgt cgacaaagag ttcaaagggc ttagtcaaat ctggcaaccc
     4801 cagggctggg gcagttagaa gagcttgctt gatttcttga taggcctttt gttggtctgg
     4861 gccccaatta aacagagtcc ccggtttggt gagagggtac aggggggctg ccatttctgc
     4921 aaacccaggg atgaagaggc gacagaagcc tgccttccct aggaactccc ttagttgtcg
     4981 aggggtcttc ggagtaggct gccccatcac agtctctttt ctggcctcag tcagccatct
     5041 ctgaccctct tttagaagat accccagata cttgacctgt ttctggcaaa tttgggcttt
     5101 cttggccgag gcccgatacc cgaggttccc tagggtttgt aacagggccc gagtaccttg
     5161 ttggcagtct agctcagaag tggcggccag cagtaagtca tccacgtact gtagcaggat
     5221 caagtctggg tgctggatcc ggaagtctgc taggtctctg tgcagtgcct cattaaacag
     5281 ggtgggactg tttttgaaac cctgtgggag tctggtccag gtcaattgtc ctgagattcc
     5341 catctctgga tctctccact caaaggcgaa gagaggctga ctggtggggt ggagtctcag
     5401 gcagaaaaag gcatccttta aatcaagcac agtgtaccac tggtgggacg gtgggagccc
     5461 gctcaagagg ttgtaagggt tgggcacggt ggggtggatg tcttccaccc gcttgttgac
     5521 ttctctcaga tcctggacag gcctataatc attagtccct ggtttcttaa cgggtagcag
     5581 gggcgtgttc cagggggact ggcagggtac cagtattccc tggtccaaca gtctctgtat
     5641 gtggggcttg atccccagtc tggcttcttg tgacatgggg tattgtttta tggacacggg
     5701 ggtagaggtt gctttcagag gtatgatcag aggagcttgg cgaactgcca gtcccatgcc
     5761 cccggtttcc gcccaggcct gaggaaaatc agacagccat gtggacccta gagaaacatc
     5821 tggctctttt gaggtctcat gtagccgata ctcatcttct atatttaggg tgctgctgcc
     5881 gccgctgctg ccgccactgc tctctggtgt tgctgactcg cttgttcctg gtgtctcgct
     5941 gccagaggat cctccgctag atcctccaga gtcacctccc agctgagaca ggtcgatccg
     6001 tgtctcgtac aggccggtga tgctctggtg gatcagggtg gcgtccagca cctctttggt
     6061 gctggtgtac ctcttccggt cgatggtggt gtcaaagtac ttgaaggcgg caggggctcc
     6121 cagattggtc agggtaaaca ggtggatgat attctcggcc tgctctctga tgggcttatc
     6181 ccggtgcttg ttgtaggcgg acagcacttt gtccagatta gcgtcggcca ggatcactct
     6241 cttggagaac tcgctgatct gctcgatgat ctcgtccagg tagtgcttgt gctgttccac
     6301 aaacagctgt ttctgctcat tatcctcggg ggagcccttc agcttctcat agtggctggc
     6361 caggtacagg aagttcacat atttggaggg cagggccagt tcgtttccct tctgcagttc
     6421 gccggcagag gccagcattc tcttccggcc gttttccagc tcgaacaggg agtacttagg
     6481 cagcttgatg atcaggtcct ttttcacttc tttgtagccc ttggcttcca gaaagtcgat
     6541 gggattcttc tcgaagctgc ttctttccat gatggtgatc cccagcagct ctttcacact
     6601 cttcagtttc ttggacttgc ccttttccac tttggccacc accagcacag aataggccac
     6661 ggtggggctg tcgaagccgc cgtacttctt agggtcccag tccttctttc tggcgatcag
     6721 cttatcgctg ttatgcgcaa taatatcgtt cgccacaaag ttatgcgggc ccggcacggt
     6781 cagatcaaac acttcttcca cgccatccgg ttcaatgctc acaatcggat cccaatacac
     6841 atcgctctgc gccagctgac gcagttccgg gcacgccgcc gcgactttcc gcttcttctt
     6901 tggtgactcg aactcgcttc cgtcggctgt ccgtttcatg gtggcaccgg tagttagcca
     6961 gagagctctg cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg
     7021 ggcggagttg ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc
     7081 attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc
     7141 attgatgtac tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta
     7201 ctgccaagta ggaaagtccc ataaggtcat gtactgggca caatgccagg cgggccattt
     7261 accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa
     7321 gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt
     7381 tactattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     7441 aagttatgta acgggtaccc tgatctagag gccgcaggaa cccctagtga tggagttggc
     7501 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     7561 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcag
//