LOCUS       sequence-229042-        6897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dsRNA expression .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6897)
  TITLE     Petr Svoboda lab unpublished
REFERENCE   2  (bases 1 to 6897)
  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"
COMMENT     Sequence Label: CAG-EGFP-Lin28IR
FEATURES             Location/Qualifiers
     source          1..6897
                     /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"
     regulatory      1725..1734
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1731..1733,1734..1736,1737..2450)
                     /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(1731..1733,1734..1736,1737..2447)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1776..1797)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2037..2056)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2384..2405
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3921..3940)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3986..4041
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4040..4059)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4400..4416)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4400..4416)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4413..4435)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4424..4440
                     /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(4448..4454,4455..4472,4473..4478))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4493..4514
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4573..4768
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4619..4754
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4774..4908
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4811..4830)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4865..4884
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4976..4993)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5147..5735)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5227..5246)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5906..6697,6698..6766))
                     /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     6529..6548
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6767..6871)
                     /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 tgttgtgctg tctcatcatt ttggcaaaga attcgccacc atggtgagca
     1741 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1801 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1861 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1921 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1981 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2041 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2101 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2161 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2221 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2281 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2341 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2401 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcactc
     2461 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcgag
     2521 atagctagct tcgcggccgc gactctagag tgcctgtact cttatgtgaa atcctcacac
     2581 atacatacag attcaagtct cccctctcac ttatgcacat actatatgta gtatctcaat
     2641 tctataaaac atgacaccct gcttaaatgt agtttgctta agagtgattt gtctaattaa
     2701 gaacttccca gttcctcagc agtagttaac atcccggccg gctgctctct gtggaccact
     2761 attcccctgt gcttgcatga ggtagacttc ccgcccgggt gaagggaaca ctgacaagta
     2821 ctaagtcttt ttccgtttct gaatcaaagg aagcttggtt tgacacttgt ttcgcttccc
     2881 gtctggcaag ggaaatatca cacagccttc tacaaaagaa cttccctagc ccagccactt
     2941 ggcaaagcct cgggttctag ggtggttcct ccccacaaaa gccttcactt gctttcagat
     3001 agagtgtggt aggagttcct ttgtctcatc ctttccttcc ctccaaggaa agacagggtg
     3061 acactgggaa ccccggcctg agacacggca gtgcaagcta cccacgcaac ccacccttac
     3121 ccccactttc tccactctgc agattgatgc ctctttcctt aaactcccca ttagccaaag
     3181 aataaccctg actcctgggc ctcaattctg ggcttctggg agcagggcag ggctgtggat
     3241 ctcttcctct tcctccctgg atccaggagt ttaaggaaag aggcatcaat ctgcagagtg
     3301 gagaaagtgg gggtaagggt gggttgcgtg ggtagcttgc actgccgtgt ctcaggccgg
     3361 ggttcccagt gtcaccctgt ctttccttgg agggaaggaa aggatgagac aaaggaactc
     3421 ctaccacact ctatctgaaa gcaagtgaag gcttttgtgg ggaggaacca ccctagaacc
     3481 cgaggctttg ccaagtggct gggctaggga agttcttttg tagaaggctg tgtgatattt
     3541 cccttgccag acgggaagcg aaacaagtgt caaaccaagc ttcctttgat tcagaaacgg
     3601 aaaaagactt agtacttgtc agtgttccct tcacccgggc gggaagtcta cctcatgcaa
     3661 gcacagggga atagtggtcc acagagagca gccggccggg atgttaacta ctgctgagga
     3721 actgggaagt tcttaattag acaaatcact cttaagcaaa ctacatttaa gcagggtgtc
     3781 atgttttata gaattgagat actacatata gtatgtgcat aagtgagagg ggagacttga
     3841 atctgtatgt atgtgtgagg atttcacata agagtacagg cactctagag cggccgcgtt
     3901 ggtaccaacc atggtgtaga tctttttccc tctgccaaaa attatgggga catcatgaag
     3961 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     4021 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     4081 cagaatgagt atttggttta gagtttggca acatatgcca tatgctggct gccatgaaca
     4141 aaggtggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt
     4201 ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt
     4261 ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc
     4321 ctgactactc ccagtcatag ctgtccctct tctcttatga agatccctcg acctgcagcc
     4381 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4441 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4501 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4561 gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     4621 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4681 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4741 cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt
     4801 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4861 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat
     4921 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4981 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5041 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5101 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5161 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5221 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5281 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5341 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5401 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5461 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5521 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5581 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5641 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5701 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5761 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5821 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5881 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5941 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6001 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6061 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6121 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6181 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6241 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6301 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6361 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6421 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6481 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6541 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6601 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6661 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6721 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6781 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6841 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
