LOCUS       sequence-231675-        5380 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 5380)
  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 5380)
  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..5380
                     /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             1558..1605
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             1627..1683
                     /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(2404..2421)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2410..2617
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2626..2766
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2626..2755
                     /label=AAV2 ITR
     rep_origin      2841..3296
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2928..2947)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3138..3159
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3313..3332)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3432..3454
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3492..3510)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3578..3682
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3683..3751,3752..4543)
                     /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(3901..3920)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4714..5302
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5203..5222
                     /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 ctcaacagac
      841 atgtggatcg agcggacggc tgatatttcc tgggaatcag acgccgagat cactgggagt
      901 tcagagagag tagatgttag attggacgac gatggtaatt ttcaactgat gaacgaccca
      961 ggggcaccct ggaaaggagg ggggggtagc ggtggtggcg gaggggtcct ttgggatact
     1021 ccgagtccta aggaatataa gaagggtgat actacgactg gggtctacag aattatgacc
     1081 cgagggttgc tgggatcata tcaggcggga gccggtgtta tggtggaagg cgttttccac
     1141 accttgtggc atacaacgaa gggagccgct ctgatgtctg gagaaggcag actcgatccg
     1201 tattggggct ctgtaaaaga ggaccgcctc tgctacggag gtccgtggaa acttcaacat
     1261 aagtggaatg gacaggatga ggtgcaaatg atagtagttg agcccggcaa gaacgtgaaa
     1321 aacgtgcaaa ctaagccagg cgttttcaag acacctgaag gagagattgg cgcagtcacc
     1381 ctcgactttc cgacgggaac gagcggttca ccgatagttg ataagaacgg ggacgtgatt
     1441 gggttgtatg gcaatggagt catcatgcct aacgggtcat atatctccgc tatagtacag
     1501 ggtgaacgaa tggatgagcc aattcctgcg ggttttgaac cggagatgct cactagtgat
     1561 tacaaagatg acgatgacaa agactacaag gatgatgatg ataagggatc cgaatccggc
     1621 agtggagcca cgaacttctc tctgttaaag caagcaggag acgtggaaga aaaccccggt
     1681 cctatgtctg aactcatcaa ggagaacatg cacatgaaac tctacatgga ggggaccgta
     1741 gacaatcatc acttcaagtg tacaagtgaa ggggagggga agccttacga agggactcaa
     1801 actatgagaa ttaaggttgt cgagggcggc ccccttccct ttgcgtttga catcctcgcc
     1861 accagtttct tgtatggttc caagacgttt ataaatcata cacagggaat acctgacttc
     1921 tttaaacaaa gttttccaga gggtttcaca tgggaaaggg ttaccactta tgaggatggc
     1981 ggagtactca ccgctaccca agatacatct ctgcaagatg gttgcctgat ttacaatgta
     2041 aaaatccgag gagttaactt tacatccaat ggtcctgtta tgcagaaaaa gacattgggg
     2101 tgggaagcat ttacagaaac gctctacccc gccgacgggg gcttggaggg ccgcaatgac
     2161 atggctctca aattggtagg tggtagtcat cttatcgcga acgcgaaaac tacctaccga
     2221 tccaagaagc ccgctaaaaa tctcaaaatg ccggtctatt acgtggacta cagactggaa
     2281 cgcaaggaag ctaacaacga gacatacgtt gagcaacatg aagtggccgt ggccagatat
     2341 tgcgatctgc cgagtaagct cgggcacaaa ttgaactaag aattcctaga gctcgctgat
     2401 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2461 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2521 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2581 gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga
     2641 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     2701 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     2761 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2821 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2881 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2941 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3001 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     3061 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3121 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3181 gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3241 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     3301 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     3361 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3421 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3481 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3541 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3601 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3661 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3721 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3781 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3841 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3901 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3961 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4021 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4081 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4141 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4201 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     4261 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4321 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4381 tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc
     4441 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4501 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4561 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4621 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4681 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4741 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4801 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4861 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4921 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4981 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5041 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5101 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5161 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5221 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5281 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5341 ttgctggcct tttgctcaca tgtagcgcgc gtgcgccaat
//
