LOCUS       sequence-6805-ex        5816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5816)
  AUTHORS   Meister G, Landthaler M, Dorsett Y, Tuschl T
  TITLE     Sequence-specific inhibition of microRNA- and siRNA-induced RNA 
            silencing.
  JOURNAL   RNA. 2004 Mar . 10(3):544-50.
  PUBMED    14970398
REFERENCE   2  (bases 1 to 5816)
  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: "RNA. 2004 
            Mar . 10(3):544-50."
COMMENT     Sequence Label: pcDNA/TO/EGFPmiR21
FEATURES             Location/Qualifiers
     source          1..5816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1002..1004,1005..1007,1008..1718)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1002..1004,1005..1007,1008..1718)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1047..1068)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1308..1327)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1655..1676
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1827..1844)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1833..2057
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2103..2531
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2190..2209)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2400..2421
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2540..2560)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2545..2874
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2725..2860
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2787..2806
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2922..2969
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2988..3362
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3492..3613
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3529..3548)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3583..3602
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3662..3678)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3662..3678)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3675..3697)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3686..3702
                     /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(3710..3716,3717..3734,3735..3740))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3755..3776
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3893..3910)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4064..4649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4144..4163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4820..5611,5612..5680))
                     /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     5443..5462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5681..5785)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggaacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc catggtgagc aagggcgagg
     1021 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1081 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1141 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1201 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1261 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1321 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1381 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1441 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1501 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1561 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     1621 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1681 ccgccgggat cactctcggc atggacgagc tgtacaagaa gctttaagaa ttctgcagat
     1741 atccagcaca gtggcggtca acatcagtct gataagctag gtacctccgc tcgagtctag
     1801 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     1861 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1921 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     1981 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2041 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2101 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2161 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2221 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2281 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2341 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2401 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2461 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2521 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2581 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2641 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2701 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2761 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     2821 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     2881 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg
     2941 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
     3001 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
     3061 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
     3121 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
     3181 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
     3241 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
     3301 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
     3361 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3421 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     3481 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3541 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3601 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3661 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3721 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3781 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3841 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3901 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3961 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4021 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4081 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4141 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4201 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4261 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4321 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4381 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4441 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4501 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4561 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tttttttgtt tgcaagcagc
     4621 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4681 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4741 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4801 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4861 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4921 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4981 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5041 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5101 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5161 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5221 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5281 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5341 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5401 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5461 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5521 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5581 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5641 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5701 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5761 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
