LOCUS       sequence-322217-        6277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector, Luciferase reporter to test activity of
            miRNA (miR-543).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6277)
  TITLE     Karreth lab plasmids
REFERENCE   2  (bases 1 to 6277)
  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: psiCHECK2-miR-543
FEATURES             Location/Qualifiers
     source          1..6277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        62..419
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        62..253
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(85..105)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      270..405
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          489..621
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     604..623
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        666..684
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      688..697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             694..1629
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    1692..1740
                     /note="synthetic polyadenylation signal"
     promoter        1749..2500
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2530..2539
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2536..4188
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /db_xref="GI:42718124"
                     /protein_id="AAS38485.1"
                     /translation="MADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     RBS             3495..3503
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    4232..4353
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4243..4262)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4297..4316
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4486..4590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4591..4659,4660..5451)
                     /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(4809..4828)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5622..6210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6111..6130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct
       61 tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
      181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
      241 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
      301 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
      361 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
      421 agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
      481 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
      541 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
      601 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
      661 gtacttaata cgactcacta taggctagcc accatggctt ccaaggtgta cgaccccgag
      721 caacgcaaac gcatgatcac tgggcctcag tggtgggctc gctgcaagca aatgaacgtg
      781 ctggactcct tcatcaacta ctatgattcc gagaagcacg ccgagaacgc cgtgattttt
      841 ctgcatggta acgctgcctc cagctacctg tggaggcacg tcgtgcctca catcgagccc
      901 gtggctagat gcatcatccc tgatctgatc ggaatgggta agtccggcaa gagcgggaat
      961 ggctcatatc gcctcctgga tcactacaag tacctcaccg cttggttcga gctgctgaac
     1021 cttccaaaga aaatcatctt tgtgggccac gactgggggg cttgtctggc ctttcactac
     1081 tcctacgagc accaagacaa gatcaaggcc atcgtccatg ctgagagtgt cgtggacgtg
     1141 atcgagtcct gggacgagtg gcctgacatc gaggaggata tcgccctgat caagagcgaa
     1201 gagggcgaga aaatggtgct tgagaataac ttcttcgtcg agaccatgct cccaagcaag
     1261 atcatgcgga aactggagcc tgaggagttc gctgcctacc tggagccatt caaggagaag
     1321 ggcgaggtta gacggcctac cctctcctgg cctcgcgaga tccctctcgt taagggaggc
     1381 aagcccgacg tcgtccagat tgtccgcaac tacaacgcct accttcgggc cagcgacgat
     1441 ctgcctaaga tgttcatcga gtccgaccct gggttctttt ccaacgctat tgtcgaggga
     1501 gctaagaagt tccctaacac cgagttcgtg aaggtgaagg gcctccactt cagccaggag
     1561 gacgctccag atgaaatggg taagtacatc aagagcttcg tggagcgcgt gctgaagaac
     1621 gagcagtaat tctaggcgat cgctcgagga attcaagaag tgcaccgcga atgtttgcgg
     1681 ccgctggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     1741 aggatctaaa tgagtcttcg gacctcgcgg gggccgctta agcggtggtt agggtttgtc
     1801 tgacgcgggg ggagggggaa ggaacgaaac actctcattc ggaggcggct cggggtttgg
     1861 tcttggtggc cacgggcacg cagaagagcg ccgcgatcct cttaagcacc cccccgccct
     1921 ccgtggaggc gggggtttgg tcggcgggtg gtaactggcg ggccgctgac tcgggcgggt
     1981 cgcgcgcccc agagtgtgac cttttcggtc tgctcgcaga cccccgggcg gcgccgccgc
     2041 ggcggcgacg ggctcgctgg gtcctaggct ccatggggac cgtatacgtg gacaggctct
     2101 ggagcatccg cacgactgcg gtgatattac cggagacctt ctgcgggacg agccgggtca
     2161 cgcggctgac gcggagcgtc cgttgggcga caaacaccag gacggggcac aggtacacta
     2221 tcttgtcacc cggaggcgcg agggactgca ggagcttcag ggagtggcgc agctgcttca
     2281 tccccgtggc ccgttgctcg cgtttgctgg cggtgtcccc ggaagaaata tatttgcatg
     2341 tctttagttc tatgatgaca caaaccccgc ccagcgtctt gtcattggcg aattcgaaca
     2401 cgcagatgca gtcggggcgg cgcggtccca ggtccacttc gcatattaag gtgacgcgtg
     2461 tggcctcgaa caccgagcga ccctgcagcg acccgcttaa aagcttggca ttccggtact
     2521 gttggtaaag ccaccatggc cgatgctaag aacattaaga agggccctgc tcccttctac
     2581 cctctggagg atggcaccgc tggcgagcag ctgcacaagg ccatgaagag gtatgccctg
     2641 gtgcctggca ccattgcctt caccgatgcc cacattgagg tggacatcac ctatgccgag
     2701 tacttcgaga tgtctgtgcg cctggccgag gccatgaaga ggtacggcct gaacaccaac
     2761 caccgcatcg tggtgtgctc tgagaactct ctgcagttct tcatgccagt gctgggcgcc
     2821 ctgttcatcg gagtggccgt ggcccctgct aacgacattt acaacgagcg cgagctgctg
     2881 aacagcatgg gcatttctca gcctaccgtg gtgttcgtgt ctaagaaggg cctgcagaag
     2941 atcctgaacg tgcagaagaa gctgcctatc atccagaaga tcatcatcat ggactctaag
     3001 accgactacc agggcttcca gagcatgtac acattcgtga catctcatct gcctcctggc
     3061 ttcaacgagt acgacttcgt gccagagtct ttcgacaggg acaaaaccat tgccctgatc
     3121 atgaacagct ctgggtctac cggcctgcct aagggcgtgg ccctgcctca tcgcaccgcc
     3181 tgtgtgcgct tctctcacgc ccgcgaccct attttcggca accagatcat ccccgacacc
     3241 gctattctga gcgtggtgcc attccaccac ggcttcggca tgttcaccac cctgggctac
     3301 ctgatttgcg gctttcgggt ggtgctgatg taccgcttcg aggaggagct gttcctgcgc
     3361 agcctgcaag actacaaaat tcagtctgcc ctgctggtgc caaccctgtt cagcttcttc
     3421 gctaagagca ccctgatcga caagtacgac ctgtctaacc tgcacgagat tgcctctggc
     3481 ggcgccccac tgtctaagga ggtgggcgaa gccgtggcca agcgctttca tctgccaggc
     3541 atccgccagg gctacggcct gaccgagaca accagcgcca ttctgattac cccagagggc
     3601 gacgacaagc ctggcgccgt gggcaaggtg gtgccattct tcgaggccaa ggtggtggac
     3661 ctggacaccg gcaagaccct gggagtgaac cagcgcggcg agctgtgtgt gcgcggccct
     3721 atgattatgt ccggctacgt gaataaccct gaggccacaa acgccctgat cgacaaggac
     3781 ggctggctgc actctggcga cattgcctac tgggacgagg acgagcactt cttcatcgtg
     3841 gaccgcctga agtctctgat caagtacaag ggctaccagg tggccccagc cgagctggag
     3901 tctatcctgc tgcagcaccc taacattttc gacgccggag tggccggcct gcccgacgac
     3961 gatgccggcg agctgcctgc cgccgtcgtc gtgctggaac acggcaagac catgaccgag
     4021 aaggagatcg tggactatgt ggccagccag gtgacaaccg ccaagaagct gcgcggcgga
     4081 gtggtgttcg tggacgaggt gcccaagggc ctgaccggca agctggacgc ccgcaagatc
     4141 cgcgagatcc tgatcaaggc taagaaaggc ggcaagatcg ccgtgtaata attctagagt
     4201 cggggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc
     4261 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4321 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4381 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4441 ggtaaaatcg ataaggatcc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4501 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4561 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4621 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4681 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4741 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4801 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4861 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4921 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4981 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5041 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5101 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5161 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5221 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5281 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5341 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5401 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5461 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5521 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5581 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5641 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5701 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5761 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5821 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5881 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5941 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6001 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6061 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6121 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6181 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6241 ctggcctttt gctggccttt tgctcacatg gctcgac
//
