LOCUS       sequence-440806-        5807 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cloning and U6-drive expression of ISDra2 TnpB reRNAs .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5807)
  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 5807)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-acceptor-2xBbsI
FEATURES             Location/Qualifiers
     source          1..5807
                     /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"
     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           1418..1485
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     polyA_signal    1525..1646
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1770..1891
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2021..3046)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(2021..3046)
                     /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(3095..3291)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(3103..3313)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3109..3244
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3163..3182)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3337..3357
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     complement(3623..3643)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3623..3641)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3662..3678)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3662..3678)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3675..3697)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3686..3702
                     /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(3710..3716,3717..3734,3735..3740))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3755..3776
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3893..3910)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4064..4652)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4144..4163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4823..5614,5615..5683))
                     /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     5446..5465
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5684..5788)
                     /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 gtggtcacgc 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 ccgtggtggc tgcgggaatc tcagacacct taaacgctca tggaggctat gtcagacctg
     1321 cttcggcggg caatggtctg cgaagtgaga atcacgcgac tttagtcgtg tgaggttcaa
     1381 gcgtcttcga gacggttgta aatgagcacg aagactcggc cggcatggtc ccagcctcct
     1441 cgctggcgcc ggctgggcaa catgcttcgg catggcgaat gggacttttt tggtaccagg
     1501 tacggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta
     1561 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     1621 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     1681 ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
     1741 tcgcgaaggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac
     1801 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     1861 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
     1921 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
     1981 catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg ctattccttt gccctcggac
     2041 gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga
     2101 cggccgcgct tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga
     2161 ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct
     2221 gatagagttg gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa
     2281 gctccggatg cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc
     2341 tccagaagaa gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt
     2401 caatgaccgc tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt
     2461 gcacgaggtg ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct
     2521 gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat
     2581 cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga
     2641 atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg
     2701 cgaccggctg cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct
     2761 gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt
     2821 ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac
     2881 catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag
     2941 cacgagattc ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga
     3001 tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttcatcacg tgctgatcag
     3061 atccgaaaat ggatatacaa gctcccggga gctttttgca aaagcctagg cctccaaaaa
     3121 agcctcctca ctacttctgg aatagctcag aggcagaggc ggcctcggcc tctgcataaa
     3181 taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg
     3241 ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
     3301 tacttctgcc tgctggggag cctggggact ttccacaccc taactgacac acattccaca
     3361 gaattcaagt gatctccaaa aaataagtac tttttgactg taaataaaat tgtaaggagt
     3421 aaaaagtact tttttttcta aaaaaatgta attaagtaaa agtaaaagta ttgattttta
     3481 attgtactca agtaaagtaa aaatccccaa aaataatact taagtacagt aatcaagtaa
     3541 aattactcaa gtactttaca cctctggttc ttgaccccct accttcagca agcccagcag
     3601 atccgagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     3661 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3721 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3781 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3841 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3901 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3961 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4021 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4081 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4141 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4201 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4261 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4321 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4381 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4441 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4501 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4561 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4621 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4681 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4741 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4801 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4861 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4921 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4981 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5041 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5101 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5161 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5221 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5281 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5341 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5401 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5461 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5521 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5581 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5641 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5701 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5761 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtg
//
