LOCUS       sequence-133324-        6436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for the analysis of miR-21 activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6436)
  AUTHORS   Shen J, Xia W, Khotskaya YB, Huo L, Nakanishi K, Lim SO, Du Y, Wang 
            Y, Chang WC, Chen CH, Hsu JL, Wu Y, Lam YC, James BP, Liu X, Liu CG,
            Patel DJ, Hung MC
  TITLE     EGFR modulates microRNA maturation in response to hypoxia through 
            phosphorylation of AGO2.
  JOURNAL   Nature. 2013 May 16;497(7449):383-7. doi: 10.1038/nature12080. Epub 
            2013 May 1.
  PUBMED    23636329
REFERENCE   2  (bases 1 to 6436)
  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/nature12080"; journalName: "Nature"; date: "2013-05-16- 
            16"; volume: "497"; issue: "7449"; pages: "383-7"
COMMENT     Sequence Label: pMIR-21 Reporter
FEATURES             Location/Qualifiers
     source          1..6436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(517..2166)
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIVPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     2082..2100
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     promoter        complement(2191..2394)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2220..2240)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2395..2698
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2819..2838)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2820..2838)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2850..2868)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2851..2868)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2888..2904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2888..2904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2901..2923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2912..2928
                     /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(2936..2942,2943..2960,2961..2966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2981..3002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3119..3136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3290..3878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3370..3389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4049..4840,4841..4909))
                     /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     4672..4691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4910..5014)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5102..5183
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5127..5146
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5413..6012)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5496..5516)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5993..6012
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(6109..6425)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6123..6258
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctagcggcc gcatacaaaa
      421 aaccaacaca cagatccaat gaaaataaaa gatcctttat taagctttag cttatcagac
      481 tgatgttgaa ctagtgcgca gctttcatca ctcgagcaat ttggactttc cgcccttctt
      541 ggcctttatg aggatctctc tgatttttct tgcgtcgagt tttccggtaa gacctttcgg
      601 tacttcgtcc acaaacacaa ctcctccgcg caactttttc gcggttgtta cttgactggc
      661 gacgtaatcc acgatctctt tttccgtcat cgtctttccg tgctccaaaa caacaacggc
      721 ggcgggaagt tcaccggcgt catcgtcggg aagacctgcc acgcccgcgt cgaagatgtt
      781 ggggtgttgt aacaatatcg attccaattc agcgggggcc acctgatatc ctttgtattt
      841 aattaaagac ttcaagcggt caactatgaa gaagtgttcg tcttcgtccc agtaagctat
      901 gtctccagaa tgtagccatc catccttgtc aatcaaggcg ttggtcgctt ccggattgtt
      961 tacataaccg gacataatca taggtcctct gacacataat tcgcctctct gattaacgcc
     1021 cagcgttttc ccggtatcca gatccacaac cttcgcttca aaaaatggaa caactttacc
     1081 gaccgcgccc ggtttatcat ccccctcggg tgtaatcaga atagctgatg tagtctcagt
     1141 gagcccatat ccttgtcgta tccctggaag atggaagcgt tttgcaaccg cttccccgac
     1201 ttctttcgaa agaggtgcgc ccccagaagc aatttcgtgt aaattagata aatcgtattt
     1261 gtcaatcaga gtgcttttgg cgaagaatga aaatagggtt ggtactagca acgcactttg
     1321 aattttgtaa tcctgaaggg atcgtaaaaa cagctcttct tcaaatctat acattaagac
     1381 gactcgaaat ccacatatca aatatccgag tgtagtaaac attccaaaac cgtgatggaa
     1441 tgggacaaca cttaaaatcg cagtatccgg aacgatttga ttgccaaaaa taggatctct
     1501 ggcatgcgag aatctgacgc aggcagttct atgcggaagg gccacaccct taggtaaccc
     1561 agtagatcca gaggaattca ttatcagtgc aattgttttg tcacgatcaa aggactctgg
     1621 tacaaaatcg tattcattaa aaccgggagg tagatgagat gtgacgaacg tgtacatcga
     1681 ctgaaatccc tggtaatccg ttttagaatc catgataata attttctgga ttattggtaa
     1741 ttttttttgc acgttcaaaa ttttttgcaa cccctttttg gaaacaaaca ctacggtagg
     1801 ctgcgaaatg ttcatactgt tgagcaattc acgttcatta taaatgtcgt tcgcgggcgc
     1861 aactgcaact ccgataaata acgcgcccaa caccggcata aagaattgaa gagagttttc
     1921 actgcatacg acgattctgt gatttgtatt cagcccatat cgtttcatag cttctgccaa
     1981 ccgaacggac atttcgaagt attccgcgta cgtgatgttc acctcgatat gtgcatctgt
     2041 aaaagcaatt gttccaggaa ccagggcgta tctcttcata gccttatgca gttgctctcc
     2101 agcggttcca tcctctagag gatagaatgg cgccgggcct ttctttatgt ttttggcgtc
     2161 ttccatggtg gatccacggt tcactaaacc agctctgctt atatagacct cccaccgtac
     2221 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     2281 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     2341 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     2401 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     2461 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     2521 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     2581 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     2641 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     2701 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactaag
     2761 atctggtacc ttgaattcat gcttctcctc cctttagtga gggtaattct ctctctctcc
     2821 ctatagtgag tcgtattaat tccttctctt ctatagtgtc acctaaatcg ttgcaattcg
     2881 taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2941 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3001 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3061 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3121 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3181 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3241 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3301 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3361 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3421 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3481 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3541 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3601 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3661 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3721 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3781 gagttggtag ctcttgatcc ggcaaaaaaa ccaccgctgg tagcggtggt ttttttgttt
     3841 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3901 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3961 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4021 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4081 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4141 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4201 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4261 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4321 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4381 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4441 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4501 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4561 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4621 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4681 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4741 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4801 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4861 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4921 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4981 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5041 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgagat
     5101 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     5161 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     5221 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg
     5281 attatgatcc tctagagtcg gtgggcctcg ggggcgggtg cggggtcggc ggggccgccc
     5341 cgggtggctt cggtcggagc catggggtcg tgcgctcctt tcggtcgggc gctgcgggtc
     5401 gtggggcggg cgtcaggcac cgggcttgcg ggtcatgcac caggtgcgcg gtccttcggg
     5461 cacctcgacg tcggcggtga cggtgaagcc gagccgctcg tagaagggga ggttgcgggg
     5521 cgcggaggtc tccaggaagg cgggcacccc ggcgcgctcg gccgcctcca ctccggggag
     5581 cacgacggcg ctgcccagac ccttgccctg gtggtcgggc gagacgccga cggtggccag
     5641 gaaccacgcg ggctccttgg gccggtgcgg cgccaggagg ccttccatct gttgctgcgc
     5701 ggccagccgg gaaccgctca actcggccat gcgcgggccg atctcggcga acaccgcccc
     5761 cgcttcgacg ctctccggcg tggtccagac cgccaccgcg gcgccgtcgt ccgcgaccca
     5821 caccttgccg atgtcgagcc cgacgcgcgt gaggaagagt tcttgcagct cggtgacccg
     5881 ctcgatgtgg cggtccgggt cgacggtgtg gcgcgtggcg gggtagtcgg cgaacgcggc
     5941 ggcgagggtg cgtacggccc gggggacgtc gtcgcgggtg gcgaggcgca ccgtgggctt
     6001 gtactcggtc atggaaggtc gtctccttgt gaggggtcag gggcgtgggt caggggatgg
     6061 tggcggcacc ggtcgtggcg gccgacctgc aggcatgcaa gctagctttt tgcaaaagcc
     6121 taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggccg aggcggcctc
     6181 ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg
     6241 cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag
     6301 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc
     6361 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     6421 acaccaatct ttcgtc
//
