LOCUS       sequence-322218-        6279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector, Luciferase reporter to test activity of
            miRNA (miR-182).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6279)
  TITLE     Karreth lab plasmids
REFERENCE   2  (bases 1 to 6279)
  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-182-5p
FEATURES             Location/Qualifiers
     source          1..6279
                     /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    1694..1742
                     /note="synthetic polyadenylation signal"
     promoter        1751..2502
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2532..2541
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2538..4190
                     /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             3497..3505
                     /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    4234..4355
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4245..4264)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4299..4318
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4488..4592
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4593..4661,4662..5453)
                     /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(4811..4830)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5624..6212
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6113..6132
                     /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 attcagtgtg agttctacca ttgccaaagc
     1681 ggccgctggc cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg
     1741 tgaggatcta aatgagtctt cggacctcgc gggggccgct taagcggtgg ttagggtttg
     1801 tctgacgcgg ggggaggggg aaggaacgaa acactctcat tcggaggcgg ctcggggttt
     1861 ggtcttggtg gccacgggca cgcagaagag cgccgcgatc ctcttaagca cccccccgcc
     1921 ctccgtggag gcgggggttt ggtcggcggg tggtaactgg cgggccgctg actcgggcgg
     1981 gtcgcgcgcc ccagagtgtg accttttcgg tctgctcgca gacccccggg cggcgccgcc
     2041 gcggcggcga cgggctcgct gggtcctagg ctccatgggg accgtatacg tggacaggct
     2101 ctggagcatc cgcacgactg cggtgatatt accggagacc ttctgcggga cgagccgggt
     2161 cacgcggctg acgcggagcg tccgttgggc gacaaacacc aggacggggc acaggtacac
     2221 tatcttgtca cccggaggcg cgagggactg caggagcttc agggagtggc gcagctgctt
     2281 catccccgtg gcccgttgct cgcgtttgct ggcggtgtcc ccggaagaaa tatatttgca
     2341 tgtctttagt tctatgatga cacaaacccc gcccagcgtc ttgtcattgg cgaattcgaa
     2401 cacgcagatg cagtcggggc ggcgcggtcc caggtccact tcgcatatta aggtgacgcg
     2461 tgtggcctcg aacaccgagc gaccctgcag cgacccgctt aaaagcttgg cattccggta
     2521 ctgttggtaa agccaccatg gccgatgcta agaacattaa gaagggccct gctcccttct
     2581 accctctgga ggatggcacc gctggcgagc agctgcacaa ggccatgaag aggtatgccc
     2641 tggtgcctgg caccattgcc ttcaccgatg cccacattga ggtggacatc acctatgccg
     2701 agtacttcga gatgtctgtg cgcctggccg aggccatgaa gaggtacggc ctgaacacca
     2761 accaccgcat cgtggtgtgc tctgagaact ctctgcagtt cttcatgcca gtgctgggcg
     2821 ccctgttcat cggagtggcc gtggcccctg ctaacgacat ttacaacgag cgcgagctgc
     2881 tgaacagcat gggcatttct cagcctaccg tggtgttcgt gtctaagaag ggcctgcaga
     2941 agatcctgaa cgtgcagaag aagctgccta tcatccagaa gatcatcatc atggactcta
     3001 agaccgacta ccagggcttc cagagcatgt acacattcgt gacatctcat ctgcctcctg
     3061 gcttcaacga gtacgacttc gtgccagagt ctttcgacag ggacaaaacc attgccctga
     3121 tcatgaacag ctctgggtct accggcctgc ctaagggcgt ggccctgcct catcgcaccg
     3181 cctgtgtgcg cttctctcac gcccgcgacc ctattttcgg caaccagatc atccccgaca
     3241 ccgctattct gagcgtggtg ccattccacc acggcttcgg catgttcacc accctgggct
     3301 acctgatttg cggctttcgg gtggtgctga tgtaccgctt cgaggaggag ctgttcctgc
     3361 gcagcctgca agactacaaa attcagtctg ccctgctggt gccaaccctg ttcagcttct
     3421 tcgctaagag caccctgatc gacaagtacg acctgtctaa cctgcacgag attgcctctg
     3481 gcggcgcccc actgtctaag gaggtgggcg aagccgtggc caagcgcttt catctgccag
     3541 gcatccgcca gggctacggc ctgaccgaga caaccagcgc cattctgatt accccagagg
     3601 gcgacgacaa gcctggcgcc gtgggcaagg tggtgccatt cttcgaggcc aaggtggtgg
     3661 acctggacac cggcaagacc ctgggagtga accagcgcgg cgagctgtgt gtgcgcggcc
     3721 ctatgattat gtccggctac gtgaataacc ctgaggccac aaacgccctg atcgacaagg
     3781 acggctggct gcactctggc gacattgcct actgggacga ggacgagcac ttcttcatcg
     3841 tggaccgcct gaagtctctg atcaagtaca agggctacca ggtggcccca gccgagctgg
     3901 agtctatcct gctgcagcac cctaacattt tcgacgccgg agtggccggc ctgcccgacg
     3961 acgatgccgg cgagctgcct gccgccgtcg tcgtgctgga acacggcaag accatgaccg
     4021 agaaggagat cgtggactat gtggccagcc aggtgacaac cgccaagaag ctgcgcggcg
     4081 gagtggtgtt cgtggacgag gtgcccaagg gcctgaccgg caagctggac gcccgcaaga
     4141 tccgcgagat cctgatcaag gctaagaaag gcggcaagat cgccgtgtaa taattctaga
     4201 gtcggggcgg ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa
     4261 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     4321 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     4381 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     4441 gtggtaaaat cgataaggat ccaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4501 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4561 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4621 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4681 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4741 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4801 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4861 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     4921 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     4981 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5041 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5101 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5161 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5221 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5281 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5341 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5401 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5461 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5521 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5581 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5641 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5701 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5761 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5821 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5881 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5941 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6001 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6061 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6121 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6181 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6241 tcctggcctt ttgctggcct tttgctcaca tggctcgac
//
