LOCUS       sequence-331324-        6900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes multiple copies of an epitope tag FLAG ( not detected by 
            anti GFP ab).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6900)
  AUTHORS   Viswanathan S, Williams ME, Bloss EB, Stasevich TJ, Speer CM, Nern 
            A, Pfeiffer BD, Hooks BM, Li WP, English BP, Tian T, Henry GL, 
            Macklin JJ, Patel R, Gerfen CR, Zhuang X, Wang Y, Rubin GM, Looger 
            LL
  TITLE     High-performance probes for light and electron microscopy.
  JOURNAL   Nat Methods. 2015 Jun;12(6):568-76. doi: 10.1038/nmeth.3365. Epub 
            2015 Apr 27.
  PUBMED    25915120
REFERENCE   2  (bases 1 to 6900)
  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"; doi: 
            "10.1038/nmeth.3365"; journalName: "Nat Methods"; date: "2015-06"; 
            volume: "12"; issue: "6"; pages: "568-76"
COMMENT     Sequence Label: pCAG_mRuby2_smFP FLAG
FEATURES             Location/Qualifiers
     source          1..6900
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          625..1641
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1565..1587
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1649..1668
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1763..1772
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1772..1795
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1799..1822
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1826..1849
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2354..2377
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2384..2407
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2420..2443
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2450..2473
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2666..2689
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2693..2716
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2720..2743
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     intron          2792..2924
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2907..2926
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2933..3521
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2986..3006)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3404..3415)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3548..3565)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3554..3665
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3761..3781)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3761..3779)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3800..3816)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3918..3937)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3983..4038
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4037..4056)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4399..4415)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4423..4439
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4447..4453,4454..4471,4472..4477))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4492..4513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4572..4767
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4618..4753
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4773..4907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4810..4829)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4864..4883
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4975..4992)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5146..5734)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5226..5245)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5905..6696,6697..6765))
                     /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     6528..6547
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6766..6870)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat
      541 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
      601 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
      661 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
      721 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt
      781 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg
      841 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc
      901 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt
      961 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg
     1021 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg
     1081 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg
     1141 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg
     1201 caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg
     1261 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt
     1321 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg
     1381 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc
     1441 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     1501 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg
     1561 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     1621 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     1681 tttggcaaag aattcggtac cgcgggcccg gcgcgccagc tggatatctc cggacccggg
     1741 ctagcgcaat tgcggccgcg ccgccaccat ggactacaag gacgacgacg acaaaggtga
     1801 ctacaaagat gatgacgata aaggcgacta taaggacgat gacgacaagg gcggaaactc
     1861 actgatcaag gaaaacatgc ggatgaaggt ggtgatggag ggctccgtga atggtcacca
     1921 gttcaagtgc accggagagg gagagggaaa cccgtacatg ggaactcaga ccatgcgcat
     1981 taaggtcatc gaaggaggtc cgctgccgtt cgctttcgat atcctggcca cttcgttcgg
     2041 aggagggtcg cgcacgttca tcaagtaccc gaagggaatc ccggacttct ttaagcagtc
     2101 attcccggaa ggattcactt gggaacgggt gacccggtat gaagatggag gtgtggtgac
     2161 tgtcatgcaa gatacttcgc tggaggatgg gtgcctcgtg taccacgtcc aagtccgcgg
     2221 agtgaatttc ccgtccaacg gaccagtgat gcagaaaaag acgaagggtt gggaacctaa
     2281 tactgaaatg atgtaccccg cagacggagg gctgaggggc tacacccaca tggcgctgaa
     2341 ggtcgacgga ggagattaca aggatgacga cgataagcaa caagattaca aagacgatga
     2401 tgacaagggc cagcagggcg actacaagga cgacgacgac aagcagcagg actacaaaga
     2461 tgacgatgat aaaggaggag gacatctgtc ctgttcgttc gtgaccacct acagatcaaa
     2521 gaaaaccgtg ggaaacatca agatgccggg cattcatgcc gtcgaccacc gcctggagcg
     2581 gctcgaagaa tcagacaatg agatgttcgt cgtgcaaaga gaacatgccg tggccaagtt
     2641 cgcgggactg ggaggcggtg gaggcgatta caaagacgat gatgacaagg gtgactataa
     2701 agacgacgat gacaaagggg attacaagga tgatgatgat aagtagaccg gtgctcgagg
     2761 taccgatgaa tagctaaggt cgaggccgca ggtaagtatc aaggttacaa gacaggttta
     2821 aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc
     2881 acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcg acaatcaacc
     2941 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3001 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3061 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3121 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3181 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3241 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3301 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     3361 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3421 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3481 ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggaattc gagctcggta
     3541 cgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3601 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3661 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3721 aagggggagg attgggaaga caatagccca gcttttgttc cctttagtga gggttaattg
     3781 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctaattc
     3841 actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct
     3901 cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc
     3961 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg
     4021 aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag
     4081 aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa
     4141 ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc
     4201 catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt
     4261 tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc
     4321 tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga cctgcagccc
     4381 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4441 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4501 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4561 ccagcggatc cgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4621 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4681 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     4741 ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt
     4801 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4861 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg
     4921 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4981 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5041 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5101 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5161 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5221 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5281 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5341 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5401 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5461 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5521 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5581 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5641 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5701 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5761 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5821 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5881 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5941 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6001 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6061 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6121 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6181 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6241 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6301 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6361 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6421 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6481 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6541 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6601 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6661 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6721 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6781 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6841 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgggtc
//
