LOCUS       Exported                7387 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  luciferase reporter for miRNA activity.
ACCESSION   .
VERSION     .
KEYWORDS    766_SMAD4 in pmiRGlo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7387)
  AUTHORS   Eichelser C, Stuckrath I, Muller V, Milde-Langosch K, Wikman H, 
            Pantel K, Schwarzenbach H
  TITLE     Increased serum levels of circulating exosomal microRNA-373 in 
            receptor-negative breast cancer patients.
  JOURNAL   Oncotarget. 2014 Oct 30;5(20):9650-63.
  PUBMED    25333260
REFERENCE   2  (bases 1 to 7387)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7387
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(60..78)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    115..236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(126..145)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     180..199
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        481..838
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(504..524)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      689..824
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     751..770
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     regulatory      883..892
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             889..1821
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             1873..2664
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1924..1943)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2534..2553
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2728..2776
                     /note="synthetic polyadenylation signal"
     promoter        2932..3036
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3037..3897
                     /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     complement(3255..3274)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4055..4643)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4135..4154)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4759..5042
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     promoter        5099..5598
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5134..5157)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5531..5550
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      5639..5648
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             5645..7297
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
ORIGIN
        1 catgcaagct gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
       61 cgctgagcaa taactagcat aaccccttgg ggcggccgct tcgagcagac atgataagat
      121 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
      181 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
      241 acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag gtttttttaa
      301 gcaagtaaaa cctctacaaa tgtggtaaaa tcgaatttta acaaaatatt aacgcttaca
      361 atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgcg
      421 gatctgcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt
      481 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
      541 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
      601 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
      661 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
      721 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
      781 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
      841 gcttgattct tctgacacaa cagtctcgaa ccaaaggctg gagccaccat ggcttccaag
      901 gtgtacgacc ccgagcaacg caaacgcatg atcactgggc ctcagtggtg ggctcgctgc
      961 aagcaaatga acgtgctgga ctccttcatc aactactatg attccgagaa gcacgccgag
     1021 aacgccgtga tttttctgca tggtaacgct gcctccagct acctgtggag gcacgtcgtg
     1081 cctcacatcg agcccgtggc tagatgcatc atccctgatc tgatcggaat gggtaagtcc
     1141 ggcaagagcg ggaatggctc atatcgcctc ctggatcact acaagtacct caccgcttgg
     1201 ttcgagctgc tgaaccttcc aaagaaaatc atctttgtgg gccacgactg gggggcttgt
     1261 ctggcctttc actactccta cgagcaccaa gacaagatca aggccatcgt ccatgctgag
     1321 agtgtcgtgg acgtgatcga gtcctgggac gagtggcctg acatcgagga ggatatcgcc
     1381 ctgatcaaga gcgaagaggg cgagaaaatg gtgcttgaga ataacttctt cgtcgagacc
     1441 atgctcccaa gcaagatcat gcggaaactg gagcctgagg agttcgctgc ctacctggag
     1501 ccattcaagg agaagggcga ggttagacgg cctaccctct cctggcctcg cgagatccct
     1561 ctcgttaagg gaggcaagcc cgacgtcgtc cagattgtcc gcaactacaa cgcctacctt
     1621 cgggccagcg acgatctgcc taagatgttc atcgagtccg accctgggtt cttttccaac
     1681 gctattgtcg agggagctaa gaagttccct aacaccgagt tcgtgaaggt gaagggcctc
     1741 cacttcagcc aggaggacgc tccagatgaa atgggtaagt acatcaagag cttcgtggag
     1801 cgcgtgctga agaacgagca gaccggtggt gggagcggag gtggcggatc aggtggcgga
     1861 ggctccggag ggattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     1921 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     1981 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     2041 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     2101 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     2161 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     2221 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     2281 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     2341 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     2401 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     2461 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     2521 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     2581 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     2641 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     2701 cgcccaacct gccatcacga tggccgcaat aaaatatctt tattttcatt acatctgtgt
     2761 gttggttttt tgtgtgaatc gatagcgata aggatcctct ttgcgcttgc gttttccctt
     2821 gtccagatag cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct
     2881 ttctacgtaa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     2941 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3001 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3061 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3121 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3181 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3241 actttcaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3301 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3361 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3421 gataacactg cggccaactt acttctgaca actatcggag gaccgaagga gctaaccgct
     3481 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3541 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3601 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3661 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3721 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3781 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3841 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaattc
     3901 gaaatgaccg accaagcgac gcccaaccgg tatcagctca ctcaaaggcg gtaatacggt
     3961 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4021 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4081 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4141 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4201 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4261 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4321 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4381 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4441 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4501 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4561 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4621 cgcgcagaaa aaaaggattt caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4681 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4741 cctagatcct tttatagtcc ggaaatacag gaacgcacgc tggatggccc ttcgctggga
     4801 tggtgaaacc atgaaaaatg gcagcttcag tggattaagt gggggtaatg tggcctgtac
     4861 cctctggttg cataggtatt catacggtta aaatttatca ggcgcgattg cggcagtttt
     4921 tcgggtggtt tgttgccatt tttacctgtc tgctgccgtg atcgcgctga acgcgtttta
     4981 gcggtgcgta caattaaggg attatggtaa atccacttac tgtctgccct cgtagccatc
     5041 gagataaacc gcagtactcc ggccacgatg cgtccggcgt agaggatcga gatctaccgg
     5101 gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc
     5161 acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc
     5221 caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg
     5281 aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt
     5341 ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg
     5401 ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt
     5461 gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc
     5521 ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
     5581 tcatctccgg gcctttcgac ctgcagccca agcttggcaa tccggtactg ttggtaaagc
     5641 caccatggaa gatgccaaaa acattaagaa gggcccagcg ccattctacc cactcgaaga
     5701 cgggaccgcc ggcgagcagc tgcacaaagc catgaagcgc tacgccctgg tgcccggcac
     5761 catcgccttt accgacgcac atatcgaggt ggacattacc tacgccgagt acttcgagat
     5821 gagcgttcgg ctggcagaag ctatgaagcg ctatgggctg aatacaaacc atcggatcgt
     5881 ggtgtgcagc gagaatagct tgcagttctt catgcccgtg ttgggtgccc tgttcatcgg
     5941 tgtggctgtg gccccagcta acgacatcta caacgagcgc gagctgctga acagcatggg
     6001 catcagccag cccaccgtcg tattcgtgag caagaaaggg ctgcaaaaga tcctcaacgt
     6061 gcaaaagaag ctaccgatca tacaaaagat catcatcatg gatagcaaga ccgactacca
     6121 gggcttccaa agcatgtaca ccttcgtgac ttcccatttg ccacccggct tcaacgagta
     6181 cgacttcgtg cccgagagct tcgaccggga caaaaccatc gccctgatca tgaacagtag
     6241 tggcagtacc ggattgccca agggcgtagc cctaccgcac cgcaccgctt gtgtccgatt
     6301 cagtcatgcc cgcgacccca tcttcggcaa ccagatcatc cccgacaccg ctatcctcag
     6361 cgtggtgcca tttcaccacg gcttcggcat gttcaccacg ctgggctact tgatctgcgg
     6421 ctttcgggtc gtgctcatgt accgcttcga ggaggagcta ttcttgcgca gcttgcaaga
     6481 ctataagatt caatctgccc tgctggtgcc cacactattt agcttcttcg ctaagagcac
     6541 tctcatcgac aagtacgacc taagcaactt gcacgagatc gccagcggcg gggcgccgct
     6601 cagcaaggag gtaggtgagg ccgtggccaa acgcttccac ctaccaggca tccgccaggg
     6661 ctacggcctg acagaaacaa ccagcgccat tctgatcacc cccgaagggg acgacaagcc
     6721 tggcgcagta ggcaaggtgg tgcccttctt cgaggctaag gtggtggact tggacaccgg
     6781 taagacactg ggtgtgaacc agcgcggcga gctgtgcgtc cgtggcccca tgatcatgag
     6841 cggctacgtt aacaaccccg aggctacaaa cgctctcatc gacaaggacg gctggctgca
     6901 cagcggcgac atcgcctact gggacgagga cgagcacttc ttcatcgtgg accggctgaa
     6961 gagcctgatc aaatacaagg gctaccaggt agccccagcc gaactggaga gcatcctgct
     7021 gcaacacccc aacatcttcg acgccggggt cgccggcctg cccgacgacg atgccggcga
     7081 gctgcccgcc gcagtcgtcg tgctggaaca cggtaaaacc atgaccgaga aggagatcgt
     7141 ggactatgtg gccagccagg ttacaaccgc caagaagctg cgcggtggtg ttgtgttcgt
     7201 ggacgaggtg cctaaaggac tgaccggcaa gttggacgcc cgcaagatcc gcgagattct
     7261 cattaaggcc aagaagggcg gcaagatcgc cgtgtaattc tagttgttta aacgagctcg
     7321 ctagctcttt cagatgtgtt tacttttgcc tggagaactt ttagctattc tagagtcgac
     7381 ctgcagg
//
