LOCUS       sequence-218328-        5335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes an CRISPR array for MbCas12a targeting superfolder GFP and 
            mRFP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5335)
  AUTHORS   Watters KE, Fellmann C, Bai HB, Ren SM, Doudna JA
  TITLE     Systematic discovery of natural CRISPR-Cas12a inhibitors.
  JOURNAL   Science. 2018 Oct 12;362(6411):236-239. doi: 
            10.1126/science.aau5138. Epub 2018 Sep 6.
  PUBMED    30190307
REFERENCE   2  (bases 1 to 5335)
  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: 
            "10.1126/science.aau5138"; journalName: "Science"; date: 
            "2018-10-12- 12"; volume: "362"; issue: "6411"; pages: "236-239"
COMMENT     Sequence Label: pKEW321-MbCas12a-RFP-GFP-array
FEATURES             Location/Qualifiers
     source          1..5335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             62..73
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             89..1168
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(108..127)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             1169..1201
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag (LVA)
                     /note="mutant LVA variant that confers accelerated 
                     degradation under some conditions (Andersen et al., 1998)"
                     /translation="AANDENYALVA"
     terminator      1249..1320
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1336..1363
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    1450..1466
                     /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)."
     protein_bind    1473..1489
                     /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)."
     CDS             complement(1741..2619)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2646..2930
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     2853..2872
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     terminator      3145..3231
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3323..3350
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      complement(3476..4064)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3556..3575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4235..5026,5027..5095))
                     /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     4858..4877
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5096..5200)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5268..5286
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(5290..5333)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattctaaa gatctggcac gtaagaggtt ccaactttca ccataatgaa acatactaga
       61 gaaagaggag aaatactaga tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
      121 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
      181 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
      241 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
      301 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
      361 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
      421 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
      481 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
      541 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
      601 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
      661 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
      721 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
      781 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
      841 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
      901 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
      961 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     1021 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     1081 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     1141 acaggtttcc cgactggaaa gcgggcaggc tgcaaacgac gaaaactacg ctttagtagc
     1201 ttaataactc tgatagtgct agtgtagatc cctactagag ccaggcatca aataaaacga
     1261 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     1321 tactagagtc acactggctc accttcgggt gggcctttct gcgtttatat attgcttaga
     1381 ataatcgatc tgcggccgca gagagtgtag cttacctagt catcgaaagc tttgctacag
     1441 cggatagaat tgtgagcgga taacaattga cattgtgagc ggataacaag atactactag
     1501 tgtctaacga ccttttaaat ttctactgtt tgtagataaa gttcgtatgg aaggttccgt
     1561 tgtctaacga ccttttaaat ttctactgtt tgtagattgc ccattaacat caccatctaa
     1621 tgtctaacga ccttttaaat ttctactgtt tgtagatcaa agcccgccga aaggcgggct
     1681 tttttttgtg gatatacctt actcgagtta gccttgatag attgtctgat tcgttaccaa
     1741 ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct
     1801 cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc
     1861 aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg
     1921 gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag
     1981 atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt
     2041 gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg
     2101 tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt
     2161 tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa
     2221 caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa
     2281 tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg
     2341 gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa
     2401 cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg
     2461 accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa
     2521 atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga
     2581 gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt
     2641 cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg tcttttactg
     2701 gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt aacaaagcgg
     2761 gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca
     2821 cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat ccataagatt
     2881 agcggatcct acctgacgct ttttatcgca actctctact gtttctccat atatcggatc
     2941 cttagtaaac ctgcaggcac tgcccatgga cctcggtacc gaatagctag ccggtaatgc
     3001 attcgctaga gctcctaaag catgcgacct gcaaccggtc tgtcacgtac gtcgccaccg
     3061 tcgacgtcgt tcgtaagtag cctagataaa taaaataatc agttaaccgc gagccccatg
     3121 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     3181 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     3241 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     3301 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
     3361 caaactctgc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3421 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3481 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3541 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3601 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3661 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3721 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3781 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3841 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3901 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3961 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4021 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4081 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4141 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4201 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4261 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4321 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4381 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4441 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4501 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4561 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4621 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4681 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4741 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4801 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4861 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4921 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4981 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5041 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5101 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5161 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5221 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     5281 cgaggcagaa tttcagataa aaaaaatcct tagctttcgc taaggatgat ttctg
//
