LOCUS       sequence-133335-        6435 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for the analysis of miR-31 activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6435)
  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 6435)
  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-31-Reporter
FEATURES             Location/Qualifiers
     source          1..6435
                     /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(516..2165)
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIVPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     2081..2099
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     promoter        complement(2190..2393)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2219..2239)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2394..2697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2818..2837)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2819..2837)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2849..2867)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2850..2867)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2887..2903)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2887..2903)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2900..2922)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2911..2927
                     /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(2935..2941,2942..2959,2960..2965))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2980..3001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3118..3135)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3289..3877)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3369..3388)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4048..4839,4840..4908))
                     /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     4671..4690
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4909..5013)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5101..5182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5126..5145
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5412..6011)
                     /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(5495..5515)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5992..6011
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(6108..6424)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6122..6257
                     /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 taagcttagg caagatgctg
      481 gcatagctac tagtgcgcag ctttcatcac tcgagcaatt tggactttcc gcccttcttg
      541 gcctttatga ggatctctct gatttttctt gcgtcgagtt ttccggtaag acctttcggt
      601 acttcgtcca caaacacaac tcctccgcgc aactttttcg cggttgttac ttgactggcg
      661 acgtaatcca cgatctcttt ttccgtcatc gtctttccgt gctccaaaac aacaacggcg
      721 gcgggaagtt caccggcgtc atcgtcggga agacctgcca cgcccgcgtc gaagatgttg
      781 gggtgttgta acaatatcga ttccaattca gcgggggcca cctgatatcc tttgtattta
      841 attaaagact tcaagcggtc aactatgaag aagtgttcgt cttcgtccca gtaagctatg
      901 tctccagaat gtagccatcc atccttgtca atcaaggcgt tggtcgcttc cggattgttt
      961 acataaccgg acataatcat aggtcctctg acacataatt cgcctctctg attaacgccc
     1021 agcgttttcc cggtatccag atccacaacc ttcgcttcaa aaaatggaac aactttaccg
     1081 accgcgcccg gtttatcatc cccctcgggt gtaatcagaa tagctgatgt agtctcagtg
     1141 agcccatatc cttgtcgtat ccctggaaga tggaagcgtt ttgcaaccgc ttccccgact
     1201 tctttcgaaa gaggtgcgcc cccagaagca atttcgtgta aattagataa atcgtatttg
     1261 tcaatcagag tgcttttggc gaagaatgaa aatagggttg gtactagcaa cgcactttga
     1321 attttgtaat cctgaaggga tcgtaaaaac agctcttctt caaatctata cattaagacg
     1381 actcgaaatc cacatatcaa atatccgagt gtagtaaaca ttccaaaacc gtgatggaat
     1441 gggacaacac ttaaaatcgc agtatccgga acgatttgat tgccaaaaat aggatctctg
     1501 gcatgcgaga atctgacgca ggcagttcta tgcggaaggg ccacaccctt aggtaaccca
     1561 gtagatccag aggaattcat tatcagtgca attgttttgt cacgatcaaa ggactctggt
     1621 acaaaatcgt attcattaaa accgggaggt agatgagatg tgacgaacgt gtacatcgac
     1681 tgaaatccct ggtaatccgt tttagaatcc atgataataa ttttctggat tattggtaat
     1741 tttttttgca cgttcaaaat tttttgcaac ccctttttgg aaacaaacac tacggtaggc
     1801 tgcgaaatgt tcatactgtt gagcaattca cgttcattat aaatgtcgtt cgcgggcgca
     1861 actgcaactc cgataaataa cgcgcccaac accggcataa agaattgaag agagttttca
     1921 ctgcatacga cgattctgtg atttgtattc agcccatatc gtttcatagc ttctgccaac
     1981 cgaacggaca tttcgaagta ttccgcgtac gtgatgttca cctcgatatg tgcatctgta
     2041 aaagcaattg ttccaggaac cagggcgtat ctcttcatag ccttatgcag ttgctctcca
     2101 gcggttccat cctctagagg atagaatggc gccgggcctt tctttatgtt tttggcgtct
     2161 tccatggtgg atccacggtt cactaaacca gctctgctta tatagacctc ccaccgtaca
     2221 cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt
     2281 tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc
     2341 cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta
     2401 atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac
     2461 tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc
     2521 atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga
     2581 cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat
     2641 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
     2701 aactccatat atgggctatg aactaatgac cccgtaattg attactatta ataactaaga
     2761 tctggtacct tgaattcatg cttctcctcc ctttagtgag ggtaattctc tctctctccc
     2821 tatagtgagt cgtattaatt ccttctcttc tatagtgtca cctaaatcgt tgcaattcgt
     2881 aatcatgtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2941 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3001 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3061 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3121 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3181 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3241 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3301 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3361 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3421 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3481 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3541 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3601 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3661 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3721 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3781 agttggtagc tcttgatccg gcaaaaaaac caccgctggt agcggtggtt tttttgtttg
     3841 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3901 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3961 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4021 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4081 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4141 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4201 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4261 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4321 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4381 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4441 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4501 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4561 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4621 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4681 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4741 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4801 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4861 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4921 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4981 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5041 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgagatt
     5101 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     5161 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     5221 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga
     5281 ttatgatcct ctagagtcgg tgggcctcgg gggcgggtgc ggggtcggcg gggccgcccc
     5341 gggtggcttc ggtcggagcc atggggtcgt gcgctccttt cggtcgggcg ctgcgggtcg
     5401 tggggcgggc gtcaggcacc gggcttgcgg gtcatgcacc aggtgcgcgg tccttcgggc
     5461 acctcgacgt cggcggtgac ggtgaagccg agccgctcgt agaaggggag gttgcggggc
     5521 gcggaggtct ccaggaaggc gggcaccccg gcgcgctcgg ccgcctccac tccggggagc
     5581 acgacggcgc tgcccagacc cttgccctgg tggtcgggcg agacgccgac ggtggccagg
     5641 aaccacgcgg gctccttggg ccggtgcggc gccaggaggc cttccatctg ttgctgcgcg
     5701 gccagccggg aaccgctcaa ctcggccatg cgcgggccga tctcggcgaa caccgccccc
     5761 gcttcgacgc tctccggcgt ggtccagacc gccaccgcgg cgccgtcgtc cgcgacccac
     5821 accttgccga tgtcgagccc gacgcgcgtg aggaagagtt cttgcagctc ggtgacccgc
     5881 tcgatgtggc ggtccgggtc gacggtgtgg cgcgtggcgg ggtagtcggc gaacgcggcg
     5941 gcgagggtgc gtacggcccg ggggacgtcg tcgcgggtgg cgaggcgcac cgtgggcttg
     6001 tactcggtca tggaaggtcg tctccttgtg aggggtcagg ggcgtgggtc aggggatggt
     6061 ggcggcaccg gtcgtggcgg ccgacctgca ggcatgcaag ctagcttttt gcaaaagcct
     6121 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga ggcggcctcg
     6181 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     6241 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     6301 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     6361 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     6421 caccaatctt tcgtc
//
