LOCUS       sequence-231662-        5368 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 5368)
  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 5368)
  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..5368
                     /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..805
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      817..826
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             832..1539
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQAGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     CDS             1546..1593
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             1615..1671
                     /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(2392..2409)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2398..2605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2614..2754
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2614..2743
                     /label=AAV2 ITR
     rep_origin      2829..3284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2916..2935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3126..3147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3301..3320)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3420..3442
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3480..3498)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3566..3670
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3671..3739,3740..4531)
                     /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(3889..3908)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4702..5290
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5191..5210
                     /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 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg
      601 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
      661 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa
      721 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca
      781 cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggccag cggagaaagt
      841 ttgtttaagg ggccgcgtga ttacaacccg atatcgagca ccatttgtca tttgacgaat
      901 gaatctgatg ggcacacaac atcgttgtat ggtattggat ttggtccctt catcattaca
      961 aacaagcact tgtttagaag aaataatgga acactgttgg tccaatcact acatggtgta
     1021 ttcaaggtca agaacaccac gactttgcaa caacacctca ttgatgggag ggacatgata
     1081 attattcgca tgcctaagga tttcccacca tttcctcaaa agctgaaatt tagagagcca
     1141 caaagggaag agcgcatatg tcttgtgaca accaacttcc aaactaagag catgtctagc
     1201 atggtgtcag acactagttg cacattccct tcatctgatg gcatattctg gaagcattgg
     1261 attcaaacca aggatgggca ggcaggcagt ccattagtat caactagaga tgggttcatt
     1321 gttggtatac actcagcatc gaatttcacc aacacaaaca attatttcac aagcgtgccg
     1381 aaaaacttca tggaattgtt gacaaatcag gaggcgcagc agtgggttag tggttggcga
     1441 ttaaatgctg actcagtatt gtgggggggc cataaagttt tcatggtgaa acctgaagag
     1501 ccttttcagc cagttaagga agcgactcaa ctcatgaata ctagtgatta caaagatgac
     1561 gatgacaaag actacaagga tgatgatgat aagggatccg aatccggcag tggagccacg
     1621 aacttctctc tgttaaagca agcaggagac gtggaagaaa accccggtcc tatgtctgaa
     1681 ctcatcaagg agaacatgca catgaaactc tacatggagg ggaccgtaga caatcatcac
     1741 ttcaagtgta caagtgaagg ggaggggaag ccttacgaag ggactcaaac tatgagaatt
     1801 aaggttgtcg agggcggccc ccttcccttt gcgtttgaca tcctcgccac cagtttcttg
     1861 tatggttcca agacgtttat aaatcataca cagggaatac ctgacttctt taaacaaagt
     1921 tttccagagg gtttcacatg ggaaagggtt accacttatg aggatggcgg agtactcacc
     1981 gctacccaag atacatctct gcaagatggt tgcctgattt acaatgtaaa aatccgagga
     2041 gttaacttta catccaatgg tcctgttatg cagaaaaaga cattggggtg ggaagcattt
     2101 acagaaacgc tctaccccgc cgacgggggc ttggagggcc gcaatgacat ggctctcaaa
     2161 ttggtaggtg gtagtcatct tatcgcgaac gcgaaaacta cctaccgatc caagaagccc
     2221 gctaaaaatc tcaaaatgcc ggtctattac gtggactaca gactggaacg caaggaagct
     2281 aacaacgaga catacgttga gcaacatgaa gtggccgtgg ccagatattg cgatctgccg
     2341 agtaagctcg ggcacaaatt gaactaagaa ttcctagagc tcgctgatca gcctcgactg
     2401 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2461 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2521 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2581 aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca
     2641 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     2701 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     2761 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     2821 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2881 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2941 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3001 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     3061 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3121 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt
     3181 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3241 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     3301 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3361 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3421 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     3481 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3541 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3601 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3661 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3721 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3781 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3841 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3901 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3961 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4021 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4081 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4141 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4201 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4261 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4321 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4381 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4441 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4501 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4561 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4621 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4681 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4741 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4801 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     4861 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4921 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4981 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5041 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5101 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5161 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5221 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5281 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5341 tgctcacatg tagcgcgcgt gcgccaat
//
