LOCUS       sequence-387957-        4321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning MmCas12m spacers. RFP flanked by MmCas12m 
            CRISPR repeats can be digested out with BbsI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4321)
  AUTHORS   Wu WY, Mohanraju P, Liao C, Adiego-Perez B, Creutzburg SCA, Makarova
            KS, Keessen K, Lindeboom TA, Khan TS, Prinsen S, Joosten R, Yan WX, 
            Migur A, Laffeber C, Scott DA, Lebbink JHG, Koonin EV, Beisel CL, 
            van der Oost J
  TITLE     The miniature CRISPR-Cas12m effector binds DNA to block 
            transcription.
  JOURNAL   Mol Cell. 2022 Dec 1;82(23):4487-4502.e7. doi: 
            10.1016/j.molcel.2022.11.003. Epub 2022 Nov 24.
  PUBMED    36427491
REFERENCE   2  (bases 1 to 4321)
  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"; doi: 
            "10.1016/j.molcel.2022.11.003"; journalName: "Mol Cell"; date: 
            "2022-12-1- 1"; volume: "82"; issue: "23"; pages: "4487-4502.e7"
COMMENT     Sequence Label: pCRISPR-RFP entry
FEATURES             Location/Qualifiers
     source          1..4321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(100..119)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      784..1239
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(871..890)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1081..1102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      1350..1938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1839..1858
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2092..2109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2124..2264
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2190..2209)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2350..2372
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2510..2529
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2624..2658
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             2878..3555
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(3717..3734)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3887..3973
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4065..4092
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4111..4202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4203..4271,4272..4321,1..742)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
       61 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
      121 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
      181 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
      241 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
      301 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
      361 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
      421 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
      481 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
      541 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
      601 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
      661 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
      721 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
      781 tttacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
      841 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      901 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      961 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     1021 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1081 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt
     1141 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1201 ttaacgcgaa ttttaacaaa atattaacgt ttacaattta aaaggatcta ggtgaagatc
     1261 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1321 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1381 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1441 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     1501 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1561 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1621 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1681 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     1741 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     1801 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     1861 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     1921 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     1981 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2041 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2101 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2161 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2221 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     2281 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     2341 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     2401 ggcagaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     2461 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     2521 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     2581 cgtagaggat ctgctcatgt ttgacagctt atcatcgatg catttgacag ctagctcagt
     2641 cctaggtata atgctagcgc tagcgaattc gagctcggta ccgtgtcata gcccagcttg
     2701 gcgggcgaag gccaagacat gtcttcccca tacgatataa gttgtaattc ggtaccccgc
     2761 ttcggcgggg ttttttcaag ttcaaatatg tatccgctca tgagacaatg tgtggggaga
     2821 ccacaacggt ttccctctag aaataatttt gtttaactat aagaaggaga tatacatatg
     2881 gcttcctccg aagatgttat caaagagttc atgcgtttca aagttcgtat ggaaggttcc
     2941 gttaacggtc acgagttcga aatcgaaggt gaaggtgaag gtcgtccgta cgaaggtaca
     3001 cagaccgcta aactgaaagt taccaaaggt ggcccgctgc cgttcgcttg ggacatcctg
     3061 tccccgcagt tccagtacgg ttccaaagct tacgttaaac acccggctga catcccggac
     3121 tacctgaaac tgtccttccc ggaaggtttc aaatgggaac gtgttatgaa cttcgaagat
     3181 ggtggtgttg ttaccgttac ccaggactcc tccctgcaag acggtgagtt catctacaaa
     3241 gttaaactgc gtggtaccaa cttcccgtcc gacggtccgg ttatgcagaa aaaaaccatg
     3301 ggttgggaag cttccaccga acgtatgtac ccggaagatg gtgctctgaa aggtgaaatc
     3361 aaaatgcgtc tgaaactgaa agacggtggt cactacgacg ctgaagttaa aaccacctac
     3421 atggctaaaa aaccggttca gctgccgggt gcttacaaaa ccgacatcaa actggacatc
     3481 acctcccaca acgaagatta caccatcgtt gaacagtacg aacgtgctga aggtcgtcac
     3541 tccaccggtg cttaaactag tataatgatg tgttatcatt gatgcgaggc gcctatacct
     3601 ccccgcttcg gcggggtttt tttgcaagtg gcacttttcg ggtcgaagac atgtgtcata
     3661 gcccagcttg gcgggcgaag gccaagacgt tttggcggat gagagaagat tttcagcctg
     3721 atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt
     3781 agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat
     3841 ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa
     3901 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     3961 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     4021 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
     4081 ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata
     4141 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4201 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4261 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4321 c
//
