LOCUS       sequence-412775-        5903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  reRNA guide targeting HEKsite_1_3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5903)
  AUTHORS   Marquart KF, Mathis N, Mollaysa A, Muller S, Kissling L, Rothgangl 
            T, Schmidheini L, Kulcsar PI, Allam A, Kaufmann MM, Matsushita M, 
            Haenggi T, Cathomen T, Kopf M, Krauthammer M, Schwank G
  TITLE     Effective genome editing with an enhanced ISDra2 TnpB system and 
            deep learning-predicted omegaRNAs.
  JOURNAL   Nat Methods. 2024 Sep 23. doi: 10.1038/s41592-024-02418-z.
  PUBMED    39313558
REFERENCE   2  (bases 1 to 5903)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2024 Sep 23. doi: 10.1038/s41592-024-02418-z."
COMMENT     Sequence Label: pKM-U6-reRNA-hsHEKsite_1_3
FEATURES             Location/Qualifiers
     source          1..5903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     356..375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(678..689,690..852)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      877..886
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(892..925)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(963..996)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     promoter        1014..1262
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1014..1034
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1185..1204
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           1514..1581
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     polyA_signal    1621..1742
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1866..1987
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2117..3142)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(2117..3142)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(3191..3387)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(3199..3409)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3205..3340
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3259..3278)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3433..3453
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(3463..3650,3651..3662)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3719..3739)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3719..3737)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3758..3774)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3758..3774)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3771..3793)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3782..3798
                     /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(join(3806..3812,3813..3830,3831..3836))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3851..3872
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3989..4006)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4160..4748)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4240..4259)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4919..5710,5711..5779))
                     /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     5542..5561
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5780..5884)
                     /gene="bla"
                     /label=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 ggcgggtgtg gkggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
      661 cggcatatgg ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt
      721 acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac
      781 ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaag gaagtaaaag
      841 taaaagcaag aaagatcgat ctcgaaggat ctggaggcca ccatggtgtc gataacttcg
      901 tatagcatac attatacgaa gttatcgata gtccagtgtg gtggaattgc ccttggccgg
      961 ccataacttc gtataggata ctttatacga agttatggta ccggcgcgca tgtgagggcc
     1021 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     1081 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     1141 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     1201 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     1261 ccgattcaag aatcccgaag tgaagaatct tgccgtccgt acatggactt gcccgaactg
     1321 tggggaaacc catgaccgag acgagaacgc tgcgctgaac attcggcgtg aagcgttggt
     1381 ggctgcggga atctcagaca ccttaaacgc tcatggaggc tatgtcagac ctgcttcggc
     1441 gggcaatggt ctgcgaagtg agaatcacgc gactttagtc gtgtgaggtt caaagaacac
     1501 ccataagaac aacggccggc atggtcccag cctcctcgct ggcgccggct gggcaacatg
     1561 cttcggcatg gcgaatggga cttttttggt accaggtacg gccgcttcga gcagacatga
     1621 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     1681 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     1741 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt
     1801 tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcgc gaaggtcgac ggtatacaga
     1861 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     1921 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     1981 acaagttggg gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc
     2041 cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga
     2101 aatctcgtag cacgtgctat tcctttgccc tcggacgagt gctggggcgt cggtttccac
     2161 tatcggcgag tacttctaca cagccatcgg tccagacggc cgcgcttctg cgggcgattt
     2221 gtgtacgccc gacagtcccg gctccggatc ggacgattgc gtcgcatcga ccctgcgccc
     2281 aagctgcatc atcgaaattg ccgtcaacca agctctgata gagttggtca agaccaatgc
     2341 ggagcatata cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt
     2401 agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg ttggcgacct
     2461 cgtattggga atccccgaac atcgcctcgc tccagtcaat gaccgctgtt atgcggccat
     2521 tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc
     2581 agtcctcggc ccaaagcatc agctcatcga gagcctgcgc gacggacgca ctgacggtgt
     2641 cgtccatcac agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac
     2701 gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg ctcgtctggc
     2761 taagatcggc cgcagcgatc gcatccatgg cctccgcgac cggctgcaga acagcgggca
     2821 gttcggtttc aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg
     2881 tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg
     2941 caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca
     3001 ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg ccctccgaga
     3061 gctgcatcag gtcggagacg ctgtcgaact tttcgatcag aaacttctcg acagacgtcg
     3121 cggtgagttc aggctttttc atcacgtgct gatcagatcc gaaaatggat atacaagctc
     3181 ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata
     3241 gctcagaggc agaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg
     3301 gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga
     3361 ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     3421 gggactttcc acaccctaac tgacacacat tccacagaat tcaagtgatc tccaaaaaat
     3481 aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa
     3541 aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat
     3601 ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc
     3661 tggttcttga ccccctacct tcagcaagcc cagcagatcc gagctccagc ttttgttccc
     3721 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3781 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3841 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3901 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3961 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4021 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4081 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4141 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4201 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4261 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4321 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4381 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4441 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4501 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4561 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4621 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4681 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4741 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4801 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4861 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4921 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4981 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5041 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5101 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5161 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5221 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5281 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5341 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5401 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5461 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5521 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5581 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5641 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5701 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5761 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5821 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5881 cgcgcacatt tccccgaaaa gtg
//
