LOCUS       sequence-169610-        6927 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses mTagBFP2 only; used as a control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6927)
  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 6927)
  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: pEJS507-pCDest2-noAcr-mTagBFP2-IRES
FEATURES             Location/Qualifiers
     source          1..6927
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     misc_feature    94..556
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(150..167)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     377..396
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    640..761
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(896..914)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(935..951)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(935..951)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(948..970)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    959..975
                     /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(983..989,990..1007,1008..1013))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1028..1049
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1166..1183)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1337..1925)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1417..1436)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2096..2887,2888..2956))
                     /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     2719..2738
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2957..3061)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3087..3542)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3224..3245)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3436..3455
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3613..4597
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        4632..4650
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4632..4649
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    4831..4965
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        5223..5602
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5603..5814
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5811..5835
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          5975..6107
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     6090..6109
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        6152..6170
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6176..6886
                     /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"
ORIGIN
        1 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
       61 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
      121 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
      181 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
      241 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
      301 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
      361 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
      421 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt
      481 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
      541 aaaaacacga tgataagctt gccacaaccc gggatcctct agagtcgacc cgggcggccg
      601 cttcccttta gtgagggtta atgcttcgag cagacatgat aagatacatt gatgagtttg
      661 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
      721 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
      781 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct
      841 acaaatgtgg taaaatccga taaggatcga tccgggcggt acccagcttt tgttcccttt
      901 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      961 gttatccgct cacaattcca cacaacatac gagccgggag cataaagtgt aaagcctggg
     1021 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1081 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1141 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1201 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1261 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1321 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1381 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1441 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1501 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1561 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1621 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1681 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1741 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1801 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1861 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1921 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1981 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2041 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2101 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2161 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2221 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2281 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2341 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2401 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2461 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2521 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2581 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2641 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2701 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     2761 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2821 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2881 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2941 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3001 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3061 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     3121 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3181 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3241 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3301 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3361 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3421 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3481 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3541 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     3601 gctattacgc cagtcgaccg ccaattcaat atggcgtata tggactcatg ccaattcaat
     3661 atggtggatc tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat
     3721 atggtggatc tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat
     3781 atggcggacc tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg
     3841 aggaggggtc ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg
     3901 gcggccatat tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg
     3961 ccaatatggc tattggccag gttcaatact atgtattggc cctatgccat atagtattcc
     4021 atatatgggt tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat
     4081 atatggggct tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata
     4141 tatggtcttt cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc
     4201 attgacgtca attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac
     4261 cacccaccat tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc
     4321 ctattgacgt caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt
     4381 aacttggcaa gtacattact attggaagta cgccagggta cattggcagt actcccattg
     4441 acgtcaatgg cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg
     4501 gggaggggca atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta
     4561 atgggaggtc tatataagca atgctcgttt agggaaccgc cattctgcct ggggacgtcg
     4621 gagcaagctt gatttaggtg acactataga atacaagcta cttgttcttt ttgcaggatc
     4681 ccatcgattc gaagccgcca ccatgttcaa acgcgctatt atcttcactt ctttcaacgg
     4741 ctttgaaaaa gtttctcgaa ctgaaaaacg ccgccttgcc aaaatcatca atgctcgagc
     4801 ctctagaact atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag
     4861 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     4921 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4981 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcggccgcg
     5041 gcgccaatgc attgggcccg gtacctcaca tggctcgaca gatcttcaat attggccatt
     5101 agccatatta ttcattggtt atatagcata aatcaatatt ggctattggc cattgcatac
     5161 gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat gaccgccatg
     5221 ttggcattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     5281 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     5341 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     5401 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     5461 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc
     5521 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt
     5581 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata
     5641 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     5701 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactgcgatc gcccgccccg
     5761 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
     5821 gtgaaccgtc agatcactag aagctttatt gcggtagttt atcacagtta aattgctaac
     5881 gcagtcagtg cttctgacac aacagtctcg aacttaagct gcagtgactc tcttaaggta
     5941 gccttgcaga agttggtcgt gaggcactgg gcaggtaagt atcaaggtta caagacaggt
     6001 ttaaggagac caatagaaac tgggcttgtc gagacagaga agactcttgc gtttctgata
     6061 ggcacctatt ggtcttactg acatccactt tgcctttctc tccacaggtg tccactccca
     6121 gttcaattac agctcttaag gctagagtac ttaatacgac tcactatagg ctagaatggt
     6181 gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     6241 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     6301 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     6361 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
     6421 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
     6481 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     6541 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
     6601 cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa
     6661 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca agaccacata
     6721 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     6781 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     6841 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattgaa cgcgtcgagc
     6901 atgcatctag ggcggccaat tccgccc
//
