LOCUS       sequence-460793-        8819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a cortisol inducible variant of MbCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8819)
  AUTHORS   Wolf B, Shehu P, Brenker L, von Bachmann A, Kroell A-S, Southern N, 
            Holderbach S, Eigenmann J, Aschenbrenner S, Mathony J, Niopek D
  TITLE     Rational engineering of allosteric protein switches by in silico 
            prediction of domain insertion sites
  JOURNAL   bioRxiv 2024.12.04.626757
REFERENCE   2  (bases 1 to 8819)
  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: "bioRxiv 
            2024.12.04.626757"
COMMENT     Sequence Label: MbCas12a_K487_GPG_GR2
FEATURES             Location/Qualifiers
     source          1..8819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..52)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        224..603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        604..807
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     758..778
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     852..871
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        852..870
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5602..5649
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5656..5736
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(5826..5845)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5891..5946
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5945..5964)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6307..6323)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6307..6323)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6320..6342)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6331..6347
                     /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(6355..6361,6362..6379,6380..6385))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6400..6421
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6480..6675
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6526..6661
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6681..6815
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6718..6737)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6772..6791
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6883..6900)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7054..7642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7134..7153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7813..8604,8605..8673))
                     /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     8436..8455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8674..8778)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
       61 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
      121 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
      181 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      241 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      301 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      361 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      421 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      481 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      541 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      601 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      661 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      721 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      781 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
      841 cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg tttaaacggg
      901 ccctctagac tcgagcggcc gccactgtgc tggatatctg cagaattcca ccacactgga
      961 ctagtggatc cgagctcggt accgcgggcc cgggatccac cggtcgccac catgctgttc
     1021 caggacttca cccacctgta ccccctgagc aagaccgtca gattcgagct gaagcccatc
     1081 ggcaagaccc tggaacacat ccacgccaag aactttctga accaggacga gacaatggcc
     1141 gacatgtacc agaaagtgaa ggccatcctg gacgactacc accgggactt tatcgccgac
     1201 atgatgggcg aagtgaagct gaccaagctg gccgagttct acgacgtgta cctgaagttc
     1261 cggaagaacc ccaaggacga cggcctgcag aagcagctta aagacctgca ggctgtgctg
     1321 cggaaagaaa tcgtgaagcc tatcggaaac ggcggcaagt acaaggccgg ctacgacaga
     1381 ctgttcggcg ccaagctgtt caaggacggc aaagagctgg gcgacctggc caagttcgtg
     1441 atcgcccagg aaggcgagag cagccctaag ctggcccacc tggcccattt cgagaagttc
     1501 agcacctact tcaccggctt ccacgacaac cgcaagaaca tgtacagcga cgaggacaag
     1561 cacaccgcca ttgcctaccg gctgatccac gagaacctgc cccggttcat cgacaacctg
     1621 cagatcctgg ccaccatcaa gcagaagcac agcgccctgt acgaccagat catcaacgag
     1681 ctgaccgcca gcggcctgga tgtgtctctg gcctctcacc tggatggcta ccacaagctg
     1741 ctgacacagg aaggcatcac cgcctacaac accctgctgg gcggaatctc tggcgaggcc
     1801 ggcagcagaa agatccaggg catcaatgaa ctgatcaaca gccaccacaa ccagcactgc
     1861 cacaagagcg agcggattgc caagctgcgg cccctgcaca agcagattct gagcgacgga
     1921 atgggcgtgt ccttcctgcc cagcaagttc gccgacgaca gcgaagtgtg ccaggccgtg
     1981 aatgagttct accggcacta cgccgacgtg ttcgccaagg tgcagagcct gttcgacggc
     2041 ttcgacgact atcagaaaga cggcatctac gtggagtaca agaacctgaa cgagctgtcc
     2101 aagcaggcct tcggcgactt cgctctgctg ggaagagtgc tggacggcta ctatgtggac
     2161 gtcgtgaacc ccgagttcaa cgagagattt gccaaggcca agaccgacaa cgccaaagcc
     2221 aagctgacaa aagagaagga caagttcatc aagggcgtgc actccctggc ttctctggaa
     2281 caggccatcg agcactacac cgccagacac gacgacgagt ctgtgcaggc cggaaagctg
     2341 ggccagtact tcaagcacgg actggccggc gtggacaacc ccatccagaa gatccacaac
     2401 aaccactcta caatcaaggg cttcctggaa agagagcggc ctgccggcga aagagccctg
     2461 cccaagatca agggtccggg gaacagcagc cagaactggc agaggttcta ccagctgacc
     2521 aagctgctgg acagcatgca cgagatggtg ggcggcctgc tccagttctg cttctacacc
     2581 ttcgtgaaca agagcctgag cgtggagttc cccgagatgc tggccgagat catcagcaac
     2641 cagctgccta agtttaatgc cggcagcgtg aagcccctgc tgttccacca gaaggggggc
     2701 ggaagcggcg gatccggcgg atctggcgga tcgggcggga gtggactgat cagcctgctg
     2761 gaggtgatcg agcccgaggt gctgtacagc ggctacgaca gcaccctgcc cgacaccagc
     2821 accaggctga tgagcaccct gaacaggctg ggcggcaggc aggtggtgag cgccgtgaag
     2881 tgggccaagg ccctgcccgg cttcaggaac ctgcacctgg acgaccagat gaccctgctg
     2941 cagtacagct ggatgagcct gatggccttc agcctgggct ggaggagcta caagcagagc
     3001 aacggcaaca tgctgtgctt cgcccccgac ctggtgatca acgaggagag gatgcagctg
     3061 ccctacatgt acgaccagtg ccagcagatg ctgaagatca gcagcgagtt cgtgaggctg
     3121 caggtgagct acgacgagta cctgtgcatg aaggtgctgc tgctgctgag caccgtgccc
     3181 aaggacggcc tgaagagcca ggccgtgttc gacgagatca ggatgaccta catcaaggag
     3241 ctgggcaagg ccatcgtgaa gagggagggc ggtccgggga gcgacaagag ccccgagatc
     3301 agacagctga aagagctgct ggacaacgcc ctgaacgtgg cccacttcgc caaactgctg
     3361 accaccaaga ccacactgca caatcaggat ggcaacttct acggcgagtt cggagccctg
     3421 tatgatgagc tggccaagat cgccaccctg tacaacaaag tgcgggacta cctgagccag
     3481 aagcccttca gcaccgagaa gtacaagctg aatttcggca accctaccct gctgaacggc
     3541 tgggacctga acaaagagaa agataacttc ggcgtgatcc tgcagaagga cggctgctac
     3601 tacctggccc tgctggataa ggcccacaag aaggtgttcg ataacgcccc caacaccggc
     3661 aagtctgtgt atcagaagat gatctataag ctgctgcctg gccccaacaa gatgctgccc
     3721 aaggtgttct tcgccaagag caacctggac tactacaacc ccagcgccga actgctggac
     3781 aaatacgccc agggcacaca caagaaaggc gacaacttca acctgaagga ctgccacgcc
     3841 ctgatcgatt tcttcaaggc cggaatcaac aagcaccccg agtggcagca cttcggcttc
     3901 aagttcagcc ccaccagcag ctaccaggac ctgagcgact tctacagaga ggtggaaccc
     3961 cagggctacc aagtgaagtt cgtggacatc aacgccgact acatcaatga gctggtggaa
     4021 cagggccagc tgtacctgtt tcagatctac aacaaggact ttagccccaa ggcccacggc
     4081 aagcccaacc tgcacaccct gtatttcaag gccctgttta gcgaggacaa cctcgtgaat
     4141 cccatctaca aactgaacgg ggaggccgag atcttctacc ggaaggcctc cctggacatg
     4201 aacgagacaa ccatccacag agccggcgag gtgctggaaa acaagaaccc tgacaaccct
     4261 aagaaacggc agttcgtgta cgacatcatc aaggacaagc ggtacaccca ggataagttc
     4321 atgctgcacg tgcccatcac catgaacttc ggagtgcagg gcatgaccat caaagagttc
     4381 aacaaaaaag tgaaccagag catccagcag tacgacgaag tgaacgtgat cggcatcgac
     4441 cggggcgagc ggcatctgct gtatctgacc gtgatcaact ccaagggcga gattctggaa
     4501 cagagatccc tgaacgacat caccaccgcc tccgccaacg gcacccagat gaccacccct
     4561 taccacaaga tcctggacaa gcgcgagatc gagcggctga acgccagagt gggatgggga
     4621 gagatcgaga caatcaaaga actgaagtcc ggctacctgt cccacgtggt gcatcagatc
     4681 tcccagctga tgctgaagta caacgccatc gtggtgctgg aagatctgaa ttttggcttc
     4741 aagaggggcc ggttcaaggt ggaaaagcag atctaccaga acttcgagaa tgccctgatc
     4801 aagaaactga accacctggt gctgaaagac aaggccgacg acgagatcgg cagctacaag
     4861 aacgccctgc agctgactaa caacttcacc gatctgaaga gtatcggcaa gcagaccggc
     4921 tttctgttct acgtgcccgc ctggaatacc agcaagatcg accccgagac aggcttcgtg
     4981 gacctgctga agcctagata cgagaatatc gcccagagcc aggccttctt cggcaagttc
     5041 gacaagatct gctacaacgc cgataggggc tacttcgagt tccacatcga ctacgccaag
     5101 ttcaatgaca aggccaaaaa cagccggcag atctggaaaa tctgcagcca cggcgataag
     5161 cgctacgtgt acgataagac cgccaaccag aacaagggcg ccaccatcgg agtgaatgtg
     5221 aacgatgagc tgaagtccct gttcacccgc taccacatca acgataagca gcccaatctc
     5281 gtgatggaca tctgccagaa caacgacaaa gagtttcaca agagcctgat gtatctgctg
     5341 aaaacactgc tggctctgcg gtacagcaac gccagctccg acgaggattt catcctgagc
     5401 cccgtggcca acgacgaggg cgtgttcttc aatagcgccc tggccgacga tacccagccc
     5461 cagaatgccg atgccaacgg cgcctaccac attgccctga agggactgtg gctgctgaac
     5521 gaactgaaga acagcgacga tctgaacaaa gtgaagctgg ccatcgacaa ccagacctgg
     5581 ctgaatttcg cccagaacag aaagaggccg gcggccacga agaaggccgg ccaggccaag
     5641 aagaagaagg gatcctaccc atacgatgtt ccagattacg cttatcccta cgacgtgcct
     5701 gattatgcat acccatatga tgtccccgac tatgcctaaa gcggccgcac tcctcaggtg
     5761 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     5821 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     5881 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     5941 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     6001 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     6061 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     6121 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     6181 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     6241 gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta
     6301 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     6361 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     6421 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg
     6481 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     6541 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     6601 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     6661 ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca
     6721 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     6781 ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg
     6841 cgcggggata ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6901 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6961 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     7021 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     7081 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     7141 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7201 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     7261 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7321 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7381 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7441 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     7501 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     7561 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     7621 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     7681 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     7741 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     7801 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7861 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7921 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7981 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     8041 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     8101 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     8161 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     8221 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     8281 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     8341 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     8401 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     8461 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     8521 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     8581 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     8641 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     8701 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     8761 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgggtcga cggatcggg
//
