LOCUS       sequence-224373-        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"
     primer_bind     51..69
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(73..116)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     RBS             180..191
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             207..1286
                     /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(226..245)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             1287..1319
                     /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      1367..1438
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1454..1481
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    1568..1584
                     /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    1591..1607
                     /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(1859..2737)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2764..3048
                     /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     2971..2990
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     terminator      3263..3349
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3441..3468
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      complement(3594..4182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3674..3693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4353..5144,5145..5213))
                     /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     4976..4995
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5214..5318)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
       61 tcacgaggca gaatttcaga taaaaaaaat ccttagcttt cgctaaggat gatttctgga
      121 attctaaaga tctggcacgt aagaggttcc aactttcacc ataatgaaac atactagaga
      181 aagaggagaa atactagatg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
      241 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
      301 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
      361 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
      421 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
      481 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
      541 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
      601 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
      661 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
      721 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
      781 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
      841 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
      901 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
      961 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     1021 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     1081 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     1141 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     1201 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     1261 aggtttcccg actggaaagc gggcaggctg caaacgacga aaactacgct ttagtagctt
     1321 aataactctg atagtgctag tgtagatccc tactagagcc aggcatcaaa taaaacgaaa
     1381 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta
     1441 ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttatatat tgcttagaat
     1501 aatcgatctg cggccgcaga gagtgtagct tacctagtca tcgaaagctt tgctacagcg
     1561 gatagaattg tgagcggata acaattgaca ttgtgagcgg ataacaagat actactagtg
     1621 tctaacgacc ttttaaattt ctactgtttg tagataaagt tcgtatggaa ggttccgttg
     1681 tctaacgacc ttttaaattt ctactgtttg tagattgccc attaacatca ccatctaatg
     1741 tctaacgacc ttttaaattt ctactgtttg tagatcaaag cccgccgaaa ggcgggcttt
     1801 tttttgtgga tataccttac tcgagttagc cttgatagat tgtctgattc gttaccaatt
     1861 atgacaactt gacggctaca tcattcactt tttcttcaca accggcacgg aactcgctcg
     1921 ggctggcccc ggtgcatttt ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa
     1981 cattgcgacc gacggtggcg ataggcatcc gggtggtgct caaaagcagc ttcgcctggc
     2041 tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc taactgctgg cggaaaagat
     2101 gtgacagacg cgacggcgac aagcaaacat gctgtgcgac gctggcgata tcaaaattgc
     2161 tgtctgccag gtgatcgctg atgtactgac aagcctcgcg tacccgatta tccatcggtg
     2221 gatggagcga ctcgttaatc gcttccatgc gccgcagtaa caattgctca agcagattta
     2281 tcgccagcag ctccgaatag cgcccttccc cttgcccggc gttaatgatt tgcccaaaca
     2341 ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata
     2401 ttgacggcca gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa acccactggt
     2461 gataccattc gcgagcctcc ggatgacgac cgtagtgatg aatctctcct ggcgggaaca
     2521 gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg atttttcacc accccctgac
     2581 cgcgaatggt gagattgaga atataacctt tcattcccag cggtcggtcg ataaaaaaat
     2641 cgagataacc gttggcctca atcggcgtta aacccgccac cagatgggca ttaaacgagt
     2701 atcccggcag caggggatca ttttgcgctt cagccatact tttcatactc ccgccattca
     2761 gagaagaaac caattgtcca tattgcatca gacattgccg tcactgcgtc ttttactggc
     2821 tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag cattctgtaa caaagcggga
     2881 ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa tcacggcaga aaagtccaca
     2941 ttgattattt gcacggcgtc acactttgct atgccatagc atttttatcc ataagattag
     3001 cggatcctac ctgacgcttt ttatcgcaac tctctactgt ttctccatat atcggatcct
     3061 tagtaaacct gcaggcactg cccatggacc tcggtaccga atagctagcc ggtaatgcat
     3121 tcgctagagc tcctaaagca tgcgacctgc aaccggtctg tcacgtacgt cgccaccgtc
     3181 gacgtcgttc gtaagtagcc tagataaata aaataatcag ttaaccgcga gccccatgcg
     3241 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     3301 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc
     3361 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
     3421 tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca
     3481 aactctgcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3541 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccac
     3601 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3661 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3721 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3781 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3841 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3901 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3961 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4021 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4081 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4141 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4201 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4261 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4321 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4381 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4441 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4501 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4561 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4621 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4681 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4741 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4801 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4861 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4921 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4981 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5041 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5101 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5161 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     5221 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     5281 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaag
//
