LOCUS       sequence-237455-        5391 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 5391)
  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 5391)
  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..5391
                     /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"
     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             1569..1616
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             1638..1694
                     /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(2415..2432)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2421..2628
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2637..2777
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2637..2766
                     /label=AAV2 ITR
     rep_origin      2852..3307
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2939..2958)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3149..3170
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3324..3343)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3443..3465
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3503..3521)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3589..3693
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3694..3762,3763..4554)
                     /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(3912..3931)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4725..5313
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5214..5233
                     /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 gggsgrggsg grgaggkgcg
      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 gcgaagtctc
      841 tcatgcgagg gctcagggac tttaatccca tagcacaaac cgtatgccga cttaaagttt
      901 cagtggaata tggagccagt gaaatgtacg gatttgggtt tggagcatac atagtagcta
      961 accaccattt gtttaggagc tataatggga gcatggaggt acaatccatg cacggcacat
     1021 tccgcgtcaa gaatcttcac agcctttccg tactgcccat caagggaagg gacatcatcc
     1081 tgatcaagat gcctaaggat tttccggtat tcccccaaaa gctgcatttt agggcgccaa
     1141 cacagaatga gcgcatctgc ctggtcggaa ccaacttcca ggagaagtat gcaagctcta
     1201 tcatcaccga aaccagtaca acgtacaata taccgggctc cacgttttgg aaacactgga
     1261 tcgaaacaga taatgggcac tgtggccttc ccgtggtgtc cactgccgat ggttgcatag
     1321 ttggcataca ttctctggct aataacgctc atactacaaa ctactattca gcattcgatg
     1381 aagattttga gtccaagtat ttgcgaacga acgaacataa cgagtgggtt aagagttggg
     1441 tctacaaccc agacactgta ctgtggggtc ctctgaaatt gaaagattct actccaaagg
     1501 gattgtttaa gaccaccaaa cttgttcaag atctgataga ccacgatgtc gtggtggaac
     1561 aaactagtga ttacaaagat gacgatgaca aagactacaa ggatgatgat gataagggat
     1621 ccgaatccgg cagtggagcc acgaacttct ctctgttaaa gcaagcagga gacgtggaag
     1681 aaaaccccgg tcctatgtct gaactcatca aggagaacat gcacatgaaa ctctacatgg
     1741 aggggaccgt agacaatcat cacttcaagt gtacaagtga aggggagggg aagccttacg
     1801 aagggactca aactatgaga attaaggttg tcgagggcgg cccccttccc tttgcgtttg
     1861 acatcctcgc caccagtttc ttgtatggtt ccaagacgtt tataaatcat acacagggaa
     1921 tacctgactt ctttaaacaa agttttccag agggtttcac atgggaaagg gttaccactt
     1981 atgaggatgg cggagtactc accgctaccc aagatacatc tctgcaagat ggttgcctga
     2041 tttacaatgt aaaaatccga ggagttaact ttacatccaa tggtcctgtt atgcagaaaa
     2101 agacattggg gtgggaagca tttacagaaa cgctctaccc cgccgacggg ggcttggagg
     2161 gccgcaatga catggctctc aaattggtag gtggtagtca tcttatcgcg aacgcgaaaa
     2221 ctacctaccg atccaagaag cccgctaaaa atctcaaaat gccggtctat tacgtggact
     2281 acagactgga acgcaaggaa gctaacaacg agacatacgt tgagcaacat gaagtggccg
     2341 tggccagata ttgcgatctg ccgagtaagc tcgggcacaa attgaactaa gaattcctag
     2401 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2461 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2521 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2581 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga
     2641 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     2701 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     2761 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2821 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     2881 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     2941 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3001 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3061 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3121 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3181 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     3241 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     3301 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     3361 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3421 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3481 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3541 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3601 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3661 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3721 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3781 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3841 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3901 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     3961 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4021 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4081 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4141 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4201 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     4261 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     4321 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     4381 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     4441 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4501 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4561 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4621 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4681 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4741 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4801 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4861 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4921 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4981 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5041 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5101 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5161 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5221 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5281 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5341 ttcctggcct tttgctggcc ttttgctcac atgtagcgcg cgtgcgccaa t
//
