LOCUS       sequence-176784-        7189 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses V5-PPP6C, includes IRES-Emerald.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7189)
  AUTHORS   Golden RJ, Chen B, Li T, Braun J, Manjunath H, Chen X, Wu J, Schmid 
            V, Chang TC, Kopp F, Ramirez-Martinez A, Tagliabracci VS, Chen ZJ, 
            Xie Y, Mendell JT
  TITLE     An Argonaute phosphorylation cycle promotes microRNA-mediated 
            silencing.
  JOURNAL   Nature. 2017 Feb 9;542(7640):197-202. doi: 10.1038/nature21025. Epub
            2017 Jan 23.
  PUBMED    28114302
REFERENCE   2  (bases 1 to 7189)
  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/nature21025"; journalName: "Nature"; date: "2017-02-9- 9"; 
            volume: "542"; issue: "7640"; pages: "197-202"
COMMENT     Sequence Label: pCAGIG_V5-PPP6C
FEATURES             Location/Qualifiers
     source          1..7189
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1736..1777
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    1787..1811
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2733..2757)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(2834..3408,3409..3411,3412..3420)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3001..3018)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3228..3247
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3421..3423,3424..3426,3427..4140)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3421..3423,3424..3426,3427..4137)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3466..3487)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3727..3746)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4074..4095
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4213..4232)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4278..4333
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4332..4351)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4692..4708)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4692..4708)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4705..4727)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4716..4732
                     /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(4740..4746,4747..4764,4765..4770))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4785..4806
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4865..5060
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4911..5046
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5066..5200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5103..5122)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5157..5176
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5268..5285)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5439..6027)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5519..5538)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6198..6989,6990..7058))
                     /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     6821..6840
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7059..7163)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attaccacca tggcaggcaa
     1741 gccaatccct aaccctctgc tgggcctgga cagcaccgaa ttgatcacaa gtttgtacaa
     1801 aaaagcaggc accatggcgc cgctagacct ggacaagtat gtggaaatag cgcggctgtg
     1861 caagtacctg ccagagaacg acctgaagcg gctatgtgac tacgtttgtg acctcctctt
     1921 agaagagtca aatgttcagc cagtatcaac accagtaaca gtgtgtggag atatccatgg
     1981 acagttttat gacctttgtg aactgttcag aactggaggt caggttcctg acacaaacta
     2041 catatttatg ggtgattttg tagacagagg ttactatagt ttggagacct tcacttacct
     2101 tcttgcatta aaggctaaat ggcctgatcg tattacactt ttgcgaggaa atcatgagag
     2161 tagacagata acacaggtct atggatttta tgatgagtgc caaaccaaat atggaaatgc
     2221 taatgcctgg agatactgta ccaaagtttt tgacatgctc acagtagcag ctttaataga
     2281 tgagcagatt ttgtgtgtcc atggtggttt atctcctgat atcaaaacac tggatcaaat
     2341 tcgaaccatc gaacggaatc aggaaattcc tcataaagga gcattttgtg atctggtttg
     2401 gtcagatcct gaagatgtgg atacctgggc tatcagtccc cgaggagcag gttggctttt
     2461 tggagcaaag gtcacaaatg agtttgttca tatcaacaac ttaaaactca tctgcagagc
     2521 acatcaacta gtgcacgaag gctataaatt tatgtttgat gagaagctgg tgacagtatg
     2581 gtctgctcct aattactgct atcgttgtgg aaatattgct tcgatcatgg tcttcaaaga
     2641 tgtaaataca agagaaccaa agttattccg ggcagttcca gattcagaac gtgttattcc
     2701 tcccagaacg acaacgccat atttccttta gaacccagct ttcttgtaca aagtggttga
     2761 tctcgaggat tggcgcgcca agatatctta attaagttaa caggcggccg ctacgtaaat
     2821 tccgcccccc ccccccctct ccctcccccc cccctaacgt tactggccga agccgcttgg
     2881 aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca
     2941 atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc
     3001 ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag
     3061 cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg
     3121 gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac
     3181 aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa
     3241 gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc
     3301 tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc
     3361 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc
     3421 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     3481 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     3541 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     3601 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     3661 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     3721 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     3781 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     3841 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     3901 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     3961 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     4021 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     4081 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     4141 caattggcat gcggccggcc gccagcacag tggtcgatcg accaatgccc tggctcacaa
     4201 ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga
     4261 gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt
     4321 tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga
     4381 gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa caaaggtggc
     4441 tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa
     4501 aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa
     4561 catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac
     4621 tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag cccaagcttg
     4681 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4741 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4801 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg
     4861 atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4921 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4981 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5041 gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata
     5101 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5161 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc
     5221 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5281 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5341 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5401 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5461 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5521 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5581 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     5641 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5701 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5761 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5821 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5881 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5941 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6001 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6061 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6121 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6181 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6241 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6301 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6361 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6421 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6481 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6541 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6601 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6661 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6721 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6781 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6841 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6901 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6961 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7021 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7081 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7141 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
