LOCUS       sequence-170083-        7121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses anti-CRISPR protein from Type I-E and mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7121)
  AUTHORS   Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, 
            Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR
  TITLE     Naturally Occurring Off-Switches for CRISPR-Cas9.
  JOURNAL   Cell. 2016 Dec 15;167(7):1829-1838.e9. doi: 
            10.1016/j.cell.2016.11.017. Epub 2016 Dec 8.
  PUBMED    27984730
REFERENCE   2  (bases 1 to 7121)
  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.cell.2016.11.017"; journalName: "Cell"; date: 
            "2016-12-15- 15"; volume: "167"; issue: "7"; pages: "1829-1838.e9"
COMMENT     Sequence Label: pEJS481-pCDest2-AcrE2-mTagBFP2-IRES
FEATURES             Location/Qualifiers
     source          1..7121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    170..304
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        562..941
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        942..1153
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1150..1174
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1314..1446
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1429..1448
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1491..1509
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1515..2225
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    join(2279..2853,2854..2856,2857..2865)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2446..2463)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2673..2692
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    2936..3057
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(3192..3210)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3231..3247)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3231..3247)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3244..3266)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3255..3271
                     /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(3279..3285,3286..3303,3304..3309))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3324..3345
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3462..3479)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3633..4221)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3713..3732)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4392..5183,5184..5252))
                     /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     5015..5034
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5253..5357)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5383..5838)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5520..5541)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5732..5751
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5909..6893
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        6928..6946
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6928..6945
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 ttcgagggcc tcgtataccg tgaaaccggg gagagtaacg gaagtggcat gttccaggcc
       61 taccaccacc tgcacggcac taaccgcacg gagacgaccg ttggctatcc gtttcatgta
      121 atggaactct agctcgagcc tctagaacta tagtgagtcg tattacgtag atccagacat
      181 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
      241 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
      301 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
      361 tttttaattc gcggccgcgg cgccaatgca ttgggcccgg tacctcacat ggctcgacag
      421 atcttcaata ttggccatta gccatattat tcattggtta tatagcataa atcaatattg
      481 gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat attggctcat
      541 gtccaatatg accgccatgt tggcattgat tattgactag ttattaatag taatcaatta
      601 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      661 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      721 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      781 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
      841 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
      901 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      961 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1021 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1081 actgcgatcg cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1141 tataagcaga gctcgtttag tgaaccgtca gatcactaga agctttattg cggtagttta
     1201 tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga acttaagctg
     1261 cagtgactct cttaaggtag ccttgcagaa gttggtcgtg aggcactggg caggtaagta
     1321 tcaaggttac aagacaggtt taaggagacc aatagaaact gggcttgtcg agacagagaa
     1381 gactcttgcg tttctgatag gcacctattg gtcttactga catccacttt gcctttctct
     1441 ccacaggtgt ccactcccag ttcaattaca gctcttaagg ctagagtact taatacgact
     1501 cactataggc tagaatggtg tctaagggcg aagagctgat taaggagaac atgcacatga
     1561 agctgtacat ggagggcacc gtggacaacc atcacttcaa gtgcacatcc gagggcgaag
     1621 gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc ggccctctcc
     1681 ccttcgcctt cgacatcctg gctactagct tcctctacgg cagcaagacc ttcatcaacc
     1741 acacccaggg catccccgac ttcttcaagc agtccttccc tgagggcttc acatgggaga
     1801 gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc agcctccagg
     1861 acggctgcct catctacaac gtcaagatca gaggggtgaa cttcacatcc aacggccctg
     1921 tgatgcagaa gaaaacactc ggctgggagg ccttcaccga gacgctgtac cccgctgacg
     1981 gcggcctgga aggcagaaac gacatggccc tgaagctcgt gggcgggagc catctgatcg
     2041 caaacgccaa gaccacatat agatccaaga aacccgctaa gaacctcaag atgcctggcg
     2101 tctactatgt ggactacaga ctggaaagaa tcaaggaggc caacaacgag acctacgtcg
     2161 agcagcacga ggtggcagtg gccagatact gcgacctccc tagcaaactg gggcacaagc
     2221 ttaattgaac gcgtcgagca tgcatctagg gcggccaatt ccgcccctct cccccccccc
     2281 cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     2341 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     2401 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     2461 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     2521 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     2581 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     2641 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     2701 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     2761 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg
     2821 acgtggtttt cctttgaaaa acacgatgat aagcttgcca caacccggga tcctctagag
     2881 tcgacccggg cggccgcttc cctttagtga gggttaatgc ttcgagcaga catgataaga
     2941 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     3001 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     3061 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa
     3121 agcaagtaaa acctctacaa atgtggtaaa atccgataag gatcgatccg ggcggtaccc
     3181 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg
     3241 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cgggagcata
     3301 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3361 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3421 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3481 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3541 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3601 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3661 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3721 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3781 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3841 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3901 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3961 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4021 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4081 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4141 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4201 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4261 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4321 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4381 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4441 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4501 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4561 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4621 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4681 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4741 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4801 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4861 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4921 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4981 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5041 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5101 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5161 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5221 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5281 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5341 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     5401 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5461 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5521 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5581 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5641 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5701 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5761 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5821 cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     5881 cggtgcgggc ctcttcgcta ttacgccagt cgaccgccaa ttcaatatgg cgtatatgga
     5941 ctcatgccaa ttcaatatgg tggatctgga cctgtgccaa ttcaatatgg cgtatatgga
     6001 ctcgtgccaa ttcaatatgg tggatctgga ccccagccaa ttcaatatgg cggacttggc
     6061 accatgccaa ttcaatatgg cggacctggc actgtgccaa ctggggaggg gtctacttgg
     6121 cacggtgcca agtttgagga ggggtcttgg ccctgtgcca agtccgccat attgaattgg
     6181 catggtgcca ataatggcgg ccatattggc tatatgccag gatcaatata taggcaatat
     6241 ccaatatggc cctatgccaa tatggctatt ggccaggttc aatactatgt attggcccta
     6301 tgccatatag tattccatat atgggttttc ctattgacgt agatagcccc tcccaatggg
     6361 cggtcccata taccatatat ggggcttcct aataccgccc atagccactc ccccattgac
     6421 gtcaatggtc tctatatatg gtctttccta ttgacgtcat atgggcggtc ctattgacgt
     6481 atatggcgcc tcccccattg acgtcaatta cggtaaatgg cccgcctggc tcaatgccca
     6541 ttgacgtcaa taggaccacc caccattgac gtcaatggga tggctcattg cccattcata
     6601 tccgttctca cgccccctat tgacgtcaat gacggtaaat ggcccacttg gcagtacatc
     6661 aatatctatt aatagtaact tggcaagtac attactattg gaagtacgcc agggtacatt
     6721 ggcagtactc ccattgacgt caatggcggt aaatggcccg cgatggctgc caagtacatc
     6781 cccattgacg tcaatgggga ggggcaatga cgcaaatggg cgttccattg acgtaaatgg
     6841 gcggtaggcg tgcctaatgg gaggtctata taagcaatgc tcgtttaggg aaccgccatt
     6901 ctgcctgggg acgtcggagc aagcttgatt taggtgacac tatagaatac aagctacttg
     6961 ttctttttgc aggatcccat cgattcgaag ccgccaccat gaatacctat ctcatcgacc
     7021 cccgcaaaaa caacgacaac tccggcgagc gcttcacggt tgacgctgtc gacattacag
     7081 ccgccgcgaa gagcgcagcc caacagattc ttggcgagga a
//
