LOCUS       pKM-U6-reRNA-mmPCSK9        5904 bp    DNA     circular UNA 10-JUL-2023
DEFINITION  Generated by Gibson/Homology Cloning (sgBbsI
            (p2Tol-U6-2xBbsI-sgRNA-HygR)  (digested by BbsI, XbaI) Fragment 3,
            230_HDV).
ACCESSION   urn.local...mz-ghp3enz
VERSION     urn.local...mz-ghp3enz
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     rep_origin      complement(7..462)
     primer_bind     complement(144..165)
                     /note="F1 origin, forward primer"
                     /standard_name="F1ori-F"
     primer_bind     356..375
                     /note="F1 origin, reverse primer"
                     /standard_name="F1ori-R"
     primer_bind     589..611
                     /note="In lacZ gene"
                     /standard_name="M13/pUC Forward"
     primer_bind     603..620
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
                     /standard_name="M13 Forward"
     primer_bind     604..620
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /standard_name="M13 fwd"
     regulatory      630..648
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /standard_name="T7 promoter"
     primer_bind     630..649
                     /note="T7 promoter, forward primer"
                     /standard_name="T7"
     regulatory      878..887
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation
                      of translation (Kozak, 1987)"
                     /standard_name="regulatory"
     protein_bind    893..926
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (GCATACAT)."
                     /standard_name="loxP"
     protein_bind    964..997
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (GGATACTT). lox2272 sites are compatible with
                     eachother, but incompatible with loxP or loxN sites (Lee
                     andSaito, 1988)."
                     /standard_name="lox2272"
     regulatory      1015..1255
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     primer_bind     1015..1035
                     /note="Human U6 promoter, forward primer"
                     /standard_name="hU6-F"
     primer_bind     1186..1205
                     /note="Human U6 promoter, forward primer"
                     /standard_name="LKO.1 5'"
     misc_feature    1264..1494
                     /standard_name="TnpB RNA backbone"
     misc_feature    1495..1514
                     /standard_name="mmPCSK9"
     misc_feature    1515..1582
                     /standard_name="HDVr"
     misc_feature    1622..1743
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     misc_feature    1867..1988
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     CDS             complement(2118..3143)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKE"
                     /standard_name="HygR"
     regulatory      complement(3192..>3436)
                     /note="Geneious type: promoter"
                     /note="SV40 enhancer and early promoter"
                     /standard_name="SV40 promoter"
     rep_origin      3206..3341
     primer_bind     complement(3260..3279)
                     /note="SV40 promoter/origin, forward primer"
                     /standard_name="SV40pro-F"
     misc_feature    3436^3437
                     /note="Geneious type: Editing History Deletion"
                     /standard_name="TGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGC
                     CTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAA
                     TTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGATC
                     CGGCTTTAGCATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCC
                     ACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAG
                     CCTGGGGACTTTCCACACC"
     regulatory      complement(3720..3738)
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     primer_bind     complement(3720..3740)
                     /note="T3 promoter, forward primer"
                     /standard_name="T3"
     primer_bind     complement(3759..3775)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /standard_name="M13 rev"
     primer_bind     complement(3759..3775)
                     /note="In lacZ gene. Also called M13-rev"
                     /standard_name="M13 Reverse"
     primer_bind     complement(3772..3794)
                     /note="In lacZ gene"
                     /standard_name="M13/pUC Reverse"
     protein_bind    3783..3799
                     /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)."
                     /standard_name="lac operator"
     regulatory      complement(3807..3837)
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    3852..3873
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     primer_bind     complement(3990..4007)
                     /note="L4440 vector, forward primer"
                     /standard_name="L4440"
     rep_origin      complement(4161..4749)
     primer_bind     complement(4241..4260)
                     /note="pBR322 origin, forward primer"
                     /standard_name="pBR322ori-F"
     CDS             complement(4920..5780)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /standard_name="AmpR"
     primer_bind     5543..5562
                     /note="Ampicillin resistance gene, reverse primer"
                     /standard_name="Amp-R"
     regulatory      complement(5781..5885)
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
ORIGIN      
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 ccggcatatg gttcttgaca gaggtgtaaa aagtactcaa aaattttact caagtgaaag
      721 tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc tagaatctta
      781 cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattaa ggaagtaaaa
      841 gtaaaagcaa gaaagatcga tctcgaagga tctggaggcc accatggtgt cgataacttc
      901 gtatagcata cattatacga agttatcgat agtccagtgt ggtggaattg cccttggccg
      961 gccataactt cgtataggat actttatacg aagttatggt accggcgcgc atgtgagggc
     1021 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     1081 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     1141 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     1201 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     1261 accgattcaa gaatcccgaa gtgaagaatc ttgccgtccg tacatggact tgcccgaact
     1321 gtggggaaac ccatgaccga gacgagaacg ctgcgctgaa cattcggcgt gaagcgttgg
     1381 tggctgcggg aatctcagac accttaaacg ctcatggagg ctatgtcaga cctgcttcgg
     1441 cgggcaatgg tctgcgaagt gagaatcacg cgactttagt cgtgtgaggt tcaagacgtc
     1501 tttggtagag aagtggccgg catggtccca gcctcctcgc tggcgccggc tgggcaacat
     1561 gcttcggcat ggcgaatggg acttttttgg taccaggtac ggccgcttcg agcagacatg
     1621 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     1681 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     1741 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat gtgggaggtt
     1801 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg cgaaggtcga cggtatacag
     1861 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     1921 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     1981 aacaagttgg ggtgggcgaa gaactccagc atgagatccc cgcgctggag gatcatccag
     2041 ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg
     2101 aaatctcgta gcacgtgcta ttcctttgcc ctcggacgag tgctggggcg tcggtttcca
     2161 ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt
     2221 tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc
     2281 caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg
     2341 cggagcatat acgcccggag ccgcggcgat cctgcaagct ccggatgcct ccgctcgaag
     2401 tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc
     2461 tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca
     2521 ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg
     2581 cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg
     2641 tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca
     2701 cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg
     2761 ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga ccggctgcag aacagcgggc
     2821 agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag
     2881 gtcaggctct cgctgaattc cccaatgtca agcacttccg gaatcgggag cgcggccgat
     2941 gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc
     3001 aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag
     3061 agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc
     3121 gcggtgagtt caggcttttt catcacgtgc tgatcagatc cgaaaatgga tatacaagct
     3181 cccgggagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat
     3241 agctcagagg cagaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg
     3301 ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg
     3361 actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
     3421 ggggactttc cacaccctaa ctgacacaca ttccacagaa ttcaagtgat ctccaaaaaa
     3481 taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt ttttctaaaa
     3541 aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt aaagtaaaaa
     3601 tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta ctttacacct
     3661 ctggttcttg accccctacc ttcagcaagc ccagcagatc cgagctccag cttttgttcc
     3721 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     3781 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3841 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3901 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3961 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4021 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4081 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4141 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4201 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4261 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4321 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4381 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4441 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4501 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4561 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     4621 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4681 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4741 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4801 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4861 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4921 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4981 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5041 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5101 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5161 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5221 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5281 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5341 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5401 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5461 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5521 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5581 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5641 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5701 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5761 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5821 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5881 ccgcgcacat ttccccgaaa agtg
//
