LOCUS       sequence-237426-        5388 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 5388)
  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 5388)
  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: pBLO44.4_PPVpro
FEATURES             Location/Qualifiers
     source          1..5388
                     /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             1566..1613
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             1635..1691
                     /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(2412..2429)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2418..2625
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2634..2774
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2634..2763
                     /label=AAV2 ITR
     rep_origin      2849..3304
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2936..2955)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3146..3167
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3321..3340)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3440..3462
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3500..3518)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3586..3690
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3691..3759,3760..4551)
                     /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(3909..3928)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4722..5310
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5211..5230
                     /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 agcaaatctc
      841 tttttagagg gttgcgcgac tataatccca tcgcgtcatc tatttgccaa ctcaataact
      901 cctctggggc acggcagagc gagatgttcg gccttggttt tggaggattg attgttacga
      961 atcaacacct tttcaagcgc aacgatggcg aactgacaat tagatcccat cacggcgagt
     1021 ttgtggtcaa agacaccaaa acgctcaagc tcctcccatg taaaggacgg gatattgtca
     1081 ttatacggct gcccaaagat ttcccaccgt tccccagaag gctccagttc aggacgccga
     1141 ccactgagga tcgcgtctgt ttgatagggt caaactttca aacgaaatca atctcttcca
     1201 ctatgtccga gactagcgct acatatcccg tggacaattc tcacttttgg aaacactgga
     1261 tcagcactaa agacggccac tgcggactcc cgatcgtatc tacacgggac ggtagtattc
     1321 tgggcctgca ttctttggca aactcaacga acacgcaaaa tttctatgcg gctttcccag
     1381 ataattttga gacaacgtac ctgtccaatc aagataacga taattggata aagcagtggc
     1441 ggtataatcc agatgaagtc tgttgggggt cactgcaact caaaagagac ataccccaat
     1501 cccctttcac catctgtaaa cttctcacgg atcttgatgg ggagtttgtc tacacccaga
     1561 ctagtgatta caaagatgac gatgacaaag actacaagga tgatgatgat aagggatccg
     1621 aatccggcag tggagccacg aacttctctc tgttaaagca agcaggagac gtggaagaaa
     1681 accccggtcc tatgtctgaa ctcatcaagg agaacatgca catgaaactc tacatggagg
     1741 ggaccgtaga caatcatcac ttcaagtgta caagtgaagg ggaggggaag ccttacgaag
     1801 ggactcaaac tatgagaatt aaggttgtcg agggcggccc ccttcccttt gcgtttgaca
     1861 tcctcgccac cagtttcttg tatggttcca agacgtttat aaatcataca cagggaatac
     1921 ctgacttctt taaacaaagt tttccagagg gtttcacatg ggaaagggtt accacttatg
     1981 aggatggcgg agtactcacc gctacccaag atacatctct gcaagatggt tgcctgattt
     2041 acaatgtaaa aatccgagga gttaacttta catccaatgg tcctgttatg cagaaaaaga
     2101 cattggggtg ggaagcattt acagaaacgc tctaccccgc cgacgggggc ttggagggcc
     2161 gcaatgacat ggctctcaaa ttggtaggtg gtagtcatct tatcgcgaac gcgaaaacta
     2221 cctaccgatc caagaagccc gctaaaaatc tcaaaatgcc ggtctattac gtggactaca
     2281 gactggaacg caaggaagct aacaacgaga catacgttga gcaacatgaa gtggccgtgg
     2341 ccagatattg cgatctgccg agtaagctcg ggcacaaatt gaactaagaa ttcctagagc
     2401 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2461 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2521 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2581 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     2641 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     2701 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     2761 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2821 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     2881 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     2941 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3001 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3061 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3121 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3181 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     3241 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     3301 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3361 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3421 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3481 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3541 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3601 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3661 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3721 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3781 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3841 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3901 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3961 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4021 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4081 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4141 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4201 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4261 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4321 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4381 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     4441 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4501 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4561 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4621 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4681 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4741 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4801 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4861 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4921 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4981 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5041 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5101 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5161 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5221 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5281 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5341 ctggcctttt gctggccttt tgctcacatg tagcgcgcgt gcgccaat
//
