LOCUS       sequence-237421-        5378 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 5378)
  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 5378)
  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: pBLO44.7_WNVpro
FEATURES             Location/Qualifiers
     source          1..5378
                     /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..575
                     /label=chicken beta-actin promoter
     intron          576..803
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      815..824
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1556..1603
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             1625..1681
                     /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(2402..2419)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2408..2615
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2624..2764
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2624..2753
                     /label=AAV2 ITR
     rep_origin      2839..3294
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2926..2945)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3136..3157
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3311..3330)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3430..3452
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3490..3508)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3576..3680
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3681..3749,3750..4541)
                     /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(3899..3918)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4712..5300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5201..5220
                     /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 gggggggggc
      421 gcgcgccmgg cggggsgggg sggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg
      481 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
      541 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
      601 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
      661 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga
      721 ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc
      781 tgcctgaaat cacttttttt caggttggac cggtgccacc atggccagct caacagacat
      841 gtggatcgag cggacggctg atatttcctg ggaatcagac gccgagatca ctgggagttc
      901 agagagagta gatgttagat tggacgacga tggtaatttt caactgatga acgacccagg
      961 ggcaccctgg aaaggagggg ggggtagcgg tggtggcgga ggggtccttt gggatactcc
     1021 gagtcctaag gaatataaga agggtgatac tacgactggg gtctacagaa ttatgacccg
     1081 agggttgctg ggatcatatc aggcgggagc cggtgttatg gtggaaggcg ttttccacac
     1141 cttgtggcat acaacgaagg gagccgctct gatgtctgga gaaggcagac tcgatccgta
     1201 ttggggctct gtaaaagagg accgcctctg ctacggaggt ccgtggaaac ttcaacataa
     1261 gtggaatgga caggatgagg tgcaaatgat agtagttgag cccggcaaga acgtgaaaaa
     1321 cgtgcaaact aagccaggcg ttttcaagac acctgaagga gagattggcg cagtcaccct
     1381 cgactttccg acgggaacga gcggttcacc gatagttgat aagaacgggg acgtgattgg
     1441 gttgtatggc aatggagtca tcatgcctaa cgggtcatat atctccgcta tagtacaggg
     1501 tgaacgaatg gatgagccaa ttcctgcggg ttttgaaccg gagatgctca ctagtgatta
     1561 caaagatgac gatgacaaag actacaagga tgatgatgat aagggatccg aatccggcag
     1621 tggagccacg aacttctctc tgttaaagca agcaggagac gtggaagaaa accccggtcc
     1681 tatgtctgaa ctcatcaagg agaacatgca catgaaactc tacatggagg ggaccgtaga
     1741 caatcatcac ttcaagtgta caagtgaagg ggaggggaag ccttacgaag ggactcaaac
     1801 tatgagaatt aaggttgtcg agggcggccc ccttcccttt gcgtttgaca tcctcgccac
     1861 cagtttcttg tatggttcca agacgtttat aaatcataca cagggaatac ctgacttctt
     1921 taaacaaagt tttccagagg gtttcacatg ggaaagggtt accacttatg aggatggcgg
     1981 agtactcacc gctacccaag atacatctct gcaagatggt tgcctgattt acaatgtaaa
     2041 aatccgagga gttaacttta catccaatgg tcctgttatg cagaaaaaga cattggggtg
     2101 ggaagcattt acagaaacgc tctaccccgc cgacgggggc ttggagggcc gcaatgacat
     2161 ggctctcaaa ttggtaggtg gtagtcatct tatcgcgaac gcgaaaacta cctaccgatc
     2221 caagaagccc gctaaaaatc tcaaaatgcc ggtctattac gtggactaca gactggaacg
     2281 caaggaagct aacaacgaga catacgttga gcaacatgaa gtggccgtgg ccagatattg
     2341 cgatctgccg agtaagctcg ggcacaaatt gaactaagaa ttcctagagc tcgctgatca
     2401 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2461 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2521 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2581 gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg
     2641 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     2701 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     2761 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     2821 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2881 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     2941 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3001 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     3061 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3121 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     3181 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     3241 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     3301 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3361 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3421 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3481 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3541 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3601 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3661 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3721 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3781 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3841 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3901 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     3961 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4021 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4081 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4141 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4201 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4261 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4321 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4381 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4441 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4501 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4561 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4621 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4681 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4741 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4801 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4861 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4921 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4981 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5041 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5101 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5161 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5221 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5281 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5341 gctggccttt tgctcacatg tagcgcgcgt gcgccaat
//
