LOCUS       sequence-231665-        5392 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 5392)
  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 5392)
  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: pBLO44.5_PVYpro
FEATURES             Location/Qualifiers
     source          1..5392
                     /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             1570..1617
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             1639..1695
                     /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(2416..2433)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2422..2629
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2638..2778
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2638..2767
                     /label=AAV2 ITR
     rep_origin      2853..3308
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2940..2959)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3150..3171
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3325..3344)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3444..3466
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3504..3522)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3590..3694
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3695..3763,3764..4555)
                     /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(3913..3932)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4726..5314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5215..5234
                     /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 cgcgaagtct
      841 ctcatgcgag ggctcaggga ctttaatccc atagcacaaa ccgtatgccg acttaaagtt
      901 tcagtggaat atggagccag tgaaatgtac ggatttgggt ttggagcata catagtagct
      961 aaccaccatt tgtttaggag ctataatggg agcatggagg tacaatccat gcacggcaca
     1021 ttccgcgtca agaatcttca cagcctttcc gtactgccca tcaagggaag ggacatcatc
     1081 ctgatcaaga tgcctaagga ttttccggta ttcccccaaa agctgcattt tagggcgcca
     1141 acacagaatg agcgcatctg cctggtcgga accaacttcc aggagaagta tgcaagctct
     1201 atcatcaccg aaaccagtac aacgtacaat ataccgggct ccacgttttg gaaacactgg
     1261 atcgaaacag ataatgggca ctgtggcctt cccgtggtgt ccactgccga tggttgcata
     1321 gttggcatac attctctggc taataacgct catactacaa actactattc agcattcgat
     1381 gaagattttg agtccaagta tttgcgaacg aacgaacata acgagtgggt taagagttgg
     1441 gtctacaacc cagacactgt actgtggggt cctctgaaat tgaaagattc tactccaaag
     1501 ggattgttta agaccaccaa acttgttcaa gatctgatag accacgatgt cgtggtggaa
     1561 caaactagtg attacaaaga tgacgatgac aaagactaca aggatgatga tgataaggga
     1621 tccgaatccg gcagtggagc cacgaacttc tctctgttaa agcaagcagg agacgtggaa
     1681 gaaaaccccg gtcctatgtc tgaactcatc aaggagaaca tgcacatgaa actctacatg
     1741 gaggggaccg tagacaatca tcacttcaag tgtacaagtg aaggggaggg gaagccttac
     1801 gaagggactc aaactatgag aattaaggtt gtcgagggcg gcccccttcc ctttgcgttt
     1861 gacatcctcg ccaccagttt cttgtatggt tccaagacgt ttataaatca tacacaggga
     1921 atacctgact tctttaaaca aagttttcca gagggtttca catgggaaag ggttaccact
     1981 tatgaggatg gcggagtact caccgctacc caagatacat ctctgcaaga tggttgcctg
     2041 atttacaatg taaaaatccg aggagttaac tttacatcca atggtcctgt tatgcagaaa
     2101 aagacattgg ggtgggaagc atttacagaa acgctctacc ccgccgacgg gggcttggag
     2161 ggccgcaatg acatggctct caaattggta ggtggtagtc atcttatcgc gaacgcgaaa
     2221 actacctacc gatccaagaa gcccgctaaa aatctcaaaa tgccggtcta ttacgtggac
     2281 tacagactgg aacgcaagga agctaacaac gagacatacg ttgagcaaca tgaagtggcc
     2341 gtggccagat attgcgatct gccgagtaag ctcgggcaca aattgaacta agaattccta
     2401 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2461 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2521 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2581 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg
     2641 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     2701 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     2761 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     2821 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     2881 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2941 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3001 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3061 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3121 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3181 caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3241 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     3301 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     3361 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3421 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3481 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     3541 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3601 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3661 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3721 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     3781 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     3841 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     3901 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     3961 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     4021 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     4081 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     4141 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     4201 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     4261 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4321 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4381 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     4441 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     4501 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     4561 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4621 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4681 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4741 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4801 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4861 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4921 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4981 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5041 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5101 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5161 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5221 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5281 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5341 gttcctggcc ttttgctggc cttttgctca catgtagcgc gcgtgcgcca at
//
