LOCUS       sequence-231664-        5389 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 5389)
  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 5389)
  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: pBLO44.4_PPVpro
FEATURES             Location/Qualifiers
     source          1..5389
                     /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             1567..1614
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             1636..1692
                     /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(2413..2430)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2419..2626
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2635..2775
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2635..2764
                     /label=AAV2 ITR
     rep_origin      2850..3305
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2937..2956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3147..3168
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3322..3341)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3441..3463
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3501..3519)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3587..3691
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3692..3760,3761..4552)
                     /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(3910..3929)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4723..5311
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5212..5231
                     /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 cagcaaatct
      841 ctttttagag ggttgcgcga ctataatccc atcgcgtcat ctatttgcca actcaataac
      901 tcctctgggg cacggcagag cgagatgttc ggccttggtt ttggaggatt gattgttacg
      961 aatcaacacc ttttcaagcg caacgatggc gaactgacaa ttagatccca tcacggcgag
     1021 tttgtggtca aagacaccaa aacgctcaag ctcctcccat gtaaaggacg ggatattgtc
     1081 attatacggc tgcccaaaga tttcccaccg ttccccagaa ggctccagtt caggacgccg
     1141 accactgagg atcgcgtctg tttgataggg tcaaactttc aaacgaaatc aatctcttcc
     1201 actatgtccg agactagcgc tacatatccc gtggacaatt ctcacttttg gaaacactgg
     1261 atcagcacta aagacggcca ctgcggactc ccgatcgtat ctacacggga cggtagtatt
     1321 ctgggcctgc attctttggc aaactcaacg aacacgcaaa atttctatgc ggctttccca
     1381 gataattttg agacaacgta cctgtccaat caagataacg ataattggat aaagcagtgg
     1441 cggtataatc cagatgaagt ctgttggggg tcactgcaac tcaaaagaga cataccccaa
     1501 tcccctttca ccatctgtaa acttctcacg gatcttgatg gggagtttgt ctacacccag
     1561 actagtgatt acaaagatga cgatgacaaa gactacaagg atgatgatga taagggatcc
     1621 gaatccggca gtggagccac gaacttctct ctgttaaagc aagcaggaga cgtggaagaa
     1681 aaccccggtc ctatgtctga actcatcaag gagaacatgc acatgaaact ctacatggag
     1741 gggaccgtag acaatcatca cttcaagtgt acaagtgaag gggaggggaa gccttacgaa
     1801 gggactcaaa ctatgagaat taaggttgtc gagggcggcc cccttccctt tgcgtttgac
     1861 atcctcgcca ccagtttctt gtatggttcc aagacgttta taaatcatac acagggaata
     1921 cctgacttct ttaaacaaag ttttccagag ggtttcacat gggaaagggt taccacttat
     1981 gaggatggcg gagtactcac cgctacccaa gatacatctc tgcaagatgg ttgcctgatt
     2041 tacaatgtaa aaatccgagg agttaacttt acatccaatg gtcctgttat gcagaaaaag
     2101 acattggggt gggaagcatt tacagaaacg ctctaccccg ccgacggggg cttggagggc
     2161 cgcaatgaca tggctctcaa attggtaggt ggtagtcatc ttatcgcgaa cgcgaaaact
     2221 acctaccgat ccaagaagcc cgctaaaaat ctcaaaatgc cggtctatta cgtggactac
     2281 agactggaac gcaaggaagc taacaacgag acatacgttg agcaacatga agtggccgtg
     2341 gccagatatt gcgatctgcc gagtaagctc gggcacaaat tgaactaaga attcctagag
     2401 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2461 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2521 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2581 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac
     2641 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     2701 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     2761 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     2821 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     2881 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2941 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3001 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3061 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3121 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3181 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3241 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     3301 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3361 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3421 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3481 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3541 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3601 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3661 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3721 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3781 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3841 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3901 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3961 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4021 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4081 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4141 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4201 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4261 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4321 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4381 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     4441 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4501 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4561 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4621 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4681 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4741 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4801 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4861 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4921 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4981 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5041 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5101 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5161 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5221 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5281 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5341 cctggccttt tgctggcctt ttgctcacat gtagcgcgcg tgcgccaat
//
