LOCUS       sequence-167153-        7031 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mTagBFP2 only; used as a control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7031)
  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 7031)
  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..7031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    234..368
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        665..1044
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1045..1256
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1253..1277
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1417..1549
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1532..1551
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1594..1612
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1618..2328
                     /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(2382..2956,2957..2959,2960..2968)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2549..2566)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2776..2795
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    3039..3160
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(3295..3313)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(3327..3345)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3366..3382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3366..3382)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3379..3401)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3390..3406
                     /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(3414..3420,3421..3438,3439..3444))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3459..3480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3597..3614)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3768..4356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3848..3867)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4527..5318,5319..5387))
                     /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     5150..5169
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5388..5492)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5518..5973)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5655..5676)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5867..5886
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6047..7031
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaagccg ccaccatgtt caaacgcgct
      121 attatcttca cttctttcaa cggctttgaa aaagtttctc gaactgaaaa acgccgcctt
      181 gccaaaatca tcaatgctcg agcctctaga actatagtga gtcgtattac gtagatccag
      241 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
      301 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
      361 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
      421 aggtttttta attcgcggcc gcggcgccaa tgcattgggc ccggtactaa ttcgcggccg
      481 cggcgccaat gcattgggcc cggtacctca catggctcga cagatcttca atattggcca
      541 ttagccatat tattcattgg ttatatagca taaatcaata ttggctattg gccattgcat
      601 acgttgtatc tatatcataa tatgtacatt tatattggct catgtccaat atgaccgcca
      661 tgttggcatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      721 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      781 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      841 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      901 catcaagtgt atcatatgcc aagtccgccc cctattgacg tcaatgacgg taaatggccc
      961 gcctggcatt atgcccagta catgacctta cgggactttc ctacttggca gtacatctac
     1021 gtattagtca tcgctattac catggtgatg cggttttggc agtacaccaa tgggcgtgga
     1081 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     1141 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactgcga tcgcccgccc
     1201 cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt
     1261 tagtgaaccg tcagatcact agaagcttta ttgcggtagt ttatcacagt taaattgcta
     1321 acgcagtcag tgcttctgac acaacagtct cgaacttaag ctgcagtgac tctcttaagg
     1381 tagccttgca gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt tacaagacag
     1441 gtttaaggag accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga
     1501 taggcaccta ttggtcttac tgacatccac tttgcctttc tctccacagg tgtccactcc
     1561 cagttcaatt acagctctta aggctagagt acttaatacg actcactata ggctagaatg
     1621 gtgtctaagg gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc
     1681 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     1741 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     1801 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     1861 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     1921 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     1981 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     2041 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     2101 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacgc caagaccaca
     2161 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     2221 agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca
     2281 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaattg aacgcgtcga
     2341 gcatgcatct agggcggcca attccgcccc tctccccccc ccccctctcc ctcccccccc
     2401 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     2461 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     2521 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     2581 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     2641 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     2701 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     2761 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     2821 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     2881 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     2941 aaaacacgat gataagcttg ccacaacccg ggatcctcta gagtcgaccc gggcggccgc
     3001 ttccctttag tgagggttaa tgcttcgagc agacatgata agatacattg atgagtttgg
     3061 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     3121 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     3181 ttttatgttt caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta
     3241 caaatgtggt aaaatccgat aaggatcgat ccgggcggta cccagctttt gttcccttta
     3301 gtgagggtta attccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     3361 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3421 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3481 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3541 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3601 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3661 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3721 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3781 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3841 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3901 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3961 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4021 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4081 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4141 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4201 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4261 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4321 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4381 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4441 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4501 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4561 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4621 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4681 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4741 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4801 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4861 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4921 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4981 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5041 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5101 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5161 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5221 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5281 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5341 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5401 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5461 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     5521 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5581 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5641 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     5701 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     5761 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     5821 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     5881 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     5941 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     6001 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagtcgacc atagccaatt
     6061 caatatggcg tatatggact catgccaatt caatatggtg gatctggacc tgtgccaatt
     6121 caatatggcg tatatggact cgtgccaatt caatatggtg gatctggacc ccagccaatt
     6181 caatatggcg gacttggcac catgccaatt caatatggcg gacttggcac tgtgccaact
     6241 ggggaggggt ctacttggca cggtgccaag tttgaggagg ggtcttggcc ctgtgccaag
     6301 tccgccatat tgaattggca tggtgccaat aatggcggcc atattggcta tatgccagga
     6361 tcaatatata ggcaatatcc aatatggccc tatgccaata tggctattgg ccaggttcaa
     6421 tactatgtat tggccctatg ccatatagta ttccatatat gggttttcct attgacgtag
     6481 atagcccctc ccaatgggcg gtcccatata ccatatatgg ggcttcctaa taccgcccat
     6541 agccactccc ccattgacgt caatggtctc tatatatggt ctttcctatt gacgtcatat
     6601 gggcggtcct attgacgtat atggcgcctc ccccattgac gtcaattacg gtaaatggcc
     6661 cgcctggctc aatgcccatt gacgtcaata ggaccaccca ccattgacgt caatgggatg
     6721 gctcattgcc cattcatatc cgttctcacg ccccctattg acgtcaatga cggtaaatgg
     6781 cccacttggc agtacatcaa tatctattaa tagtaacttg gcaagtacat tactattgga
     6841 aggacgccag ggtacattgg cagtactccc attgacgtca atggcggtaa atggcccgcg
     6901 atggctgcca agtacatccc cattgacgtc aatggggagg ggcaatgacg caaatgggcg
     6961 ttccattgac gtaaatgggc ggtaggcgtg cctaatggga ggtctatata agcaatgctc
     7021 gtttagggaa c
//
