LOCUS       sequence-237427-        5367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-intron_Pro_P2A-mTagBFP2_AmpR_ColE1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5367)
  AUTHORS   Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, 
            Savage DF
  TITLE     Profiling of engineering hotspots identifies an allosteric 
            CRISPR-Cas9 switch.
  JOURNAL   Nat Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528.
  PUBMED    27136077
REFERENCE   2  (bases 1 to 5367)
  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"; journalName: "Nat 
            Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528."
COMMENT     Sequence Label: pBLO44.1_dProtease
FEATURES             Location/Qualifiers
     source          1..5367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        299..576
                     /label=chicken beta-actin promoter
     intron          577..804
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      816..825
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             831..1538
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQAGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     CDS             1545..1592
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             1614..1670
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(2391..2408)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2397..2604
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2613..2753
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2613..2742
                     /label=AAV2 ITR
     rep_origin      2828..3283
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2915..2934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3125..3146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3300..3319)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3419..3441
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3479..3497)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3565..3669
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3670..3738,3739..4530)
                     /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     complement(3888..3907)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4701..5289
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5190..5209
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      421 cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg gggsgrggcg gagaggtgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      601 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      661 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
      721 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
      781 ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggccagc ggagaaagtt
      841 tgtttaaggg gccgcgtgat tacaacccga tatcgagcac catttgtcat ttgacgaatg
      901 aatctgatgg gcacacaaca tcgttgtatg gtattggatt tggtcccttc atcattacaa
      961 acaagcactt gtttagaaga aataatggaa cactgttggt ccaatcacta catggtgtat
     1021 tcaaggtcaa gaacaccacg actttgcaac aacacctcat tgatgggagg gacatgataa
     1081 ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa gctgaaattt agagagccac
     1141 aaagggaaga gcgcatatgt cttgtgacaa ccaacttcca aactaagagc atgtctagca
     1201 tggtgtcaga cactagttgc acattccctt catctgatgg catattctgg aagcattgga
     1261 ttcaaaccaa ggatgggcag gcaggcagtc cattagtatc aactagagat gggttcattg
     1321 ttggtataca ctcagcatcg aatttcacca acacaaacaa ttatttcaca agcgtgccga
     1381 aaaacttcat ggaattgttg acaaatcagg aggcgcagca gtgggttagt ggttggcgat
     1441 taaatgctga ctcagtattg tgggggggcc ataaagtttt catggtgaaa cctgaagagc
     1501 cttttcagcc agttaaggaa gcgactcaac tcatgaatac tagtgattac aaagatgacg
     1561 atgacaaaga ctacaaggat gatgatgata agggatccga atccggcagt ggagccacga
     1621 acttctctct gttaaagcaa gcaggagacg tggaagaaaa ccccggtcct atgtctgaac
     1681 tcatcaagga gaacatgcac atgaaactct acatggaggg gaccgtagac aatcatcact
     1741 tcaagtgtac aagtgaaggg gaggggaagc cttacgaagg gactcaaact atgagaatta
     1801 aggttgtcga gggcggcccc cttccctttg cgtttgacat cctcgccacc agtttcttgt
     1861 atggttccaa gacgtttata aatcatacac agggaatacc tgacttcttt aaacaaagtt
     1921 ttccagaggg tttcacatgg gaaagggtta ccacttatga ggatggcgga gtactcaccg
     1981 ctacccaaga tacatctctg caagatggtt gcctgattta caatgtaaaa atccgaggag
     2041 ttaactttac atccaatggt cctgttatgc agaaaaagac attggggtgg gaagcattta
     2101 cagaaacgct ctaccccgcc gacgggggct tggagggccg caatgacatg gctctcaaat
     2161 tggtaggtgg tagtcatctt atcgcgaacg cgaaaactac ctaccgatcc aagaagcccg
     2221 ctaaaaatct caaaatgccg gtctattacg tggactacag actggaacgc aaggaagcta
     2281 acaacgagac atacgttgag caacatgaag tggccgtggc cagatattgc gatctgccga
     2341 gtaagctcgg gcacaaattg aactaagaat tcctagagct cgctgatcag cctcgactgt
     2401 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2461 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2521 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2581 agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg agttggccac
     2641 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     2701 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     2761 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     2821 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2881 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     2941 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3001 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     3061 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3121 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     3181 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     3241 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     3301 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     3361 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3421 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     3481 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     3541 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3601 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3661 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3721 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3781 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3841 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3901 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3961 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     4021 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     4081 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     4141 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     4201 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     4261 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     4321 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     4381 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     4441 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     4501 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     4561 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4621 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4681 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4741 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4801 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     4861 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4921 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4981 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5041 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5101 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5161 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5221 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5281 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5341 gctcacatgt agcgcgcgtg cgccaat
//
