LOCUS       sequence-322216-        6276 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector, Luciferase reporter to test activity of
            miRNA (miR-653).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6276)
  TITLE     Karreth lab plasmids
REFERENCE   2  (bases 1 to 6276)
  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-653
FEATURES             Location/Qualifiers
     source          1..6276
                     /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    1691..1739
                     /note="synthetic polyadenylation signal"
     promoter        1748..2499
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2529..2538
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2535..4187
                     /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             3494..3502
                     /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    4231..4352
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4242..4261)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4296..4315
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4485..4589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4590..4658,4659..5450)
                     /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(4808..4827)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5621..6209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6110..6129
                     /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 attccagtag agattgtttc aacacgcggc
     1681 cgctggccgc aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga
     1741 ggatctaaat gagtcttcgg acctcgcggg ggccgcttaa gcggtggtta gggtttgtct
     1801 gacgcggggg gagggggaag gaacgaaaca ctctcattcg gaggcggctc ggggtttggt
     1861 cttggtggcc acgggcacgc agaagagcgc cgcgatcctc ttaagcaccc ccccgccctc
     1921 cgtggaggcg ggggtttggt cggcgggtgg taactggcgg gccgctgact cgggcgggtc
     1981 gcgcgcccca gagtgtgacc ttttcggtct gctcgcagac ccccgggcgg cgccgccgcg
     2041 gcggcgacgg gctcgctggg tcctaggctc catggggacc gtatacgtgg acaggctctg
     2101 gagcatccgc acgactgcgg tgatattacc ggagaccttc tgcgggacga gccgggtcac
     2161 gcggctgacg cggagcgtcc gttgggcgac aaacaccagg acggggcaca ggtacactat
     2221 cttgtcaccc ggaggcgcga gggactgcag gagcttcagg gagtggcgca gctgcttcat
     2281 ccccgtggcc cgttgctcgc gtttgctggc ggtgtccccg gaagaaatat atttgcatgt
     2341 ctttagttct atgatgacac aaaccccgcc cagcgtcttg tcattggcga attcgaacac
     2401 gcagatgcag tcggggcggc gcggtcccag gtccacttcg catattaagg tgacgcgtgt
     2461 ggcctcgaac accgagcgac cctgcagcga cccgcttaaa agcttggcat tccggtactg
     2521 ttggtaaagc caccatggcc gatgctaaga acattaagaa gggccctgct cccttctacc
     2581 ctctggagga tggcaccgct ggcgagcagc tgcacaaggc catgaagagg tatgccctgg
     2641 tgcctggcac cattgccttc accgatgccc acattgaggt ggacatcacc tatgccgagt
     2701 acttcgagat gtctgtgcgc ctggccgagg ccatgaagag gtacggcctg aacaccaacc
     2761 accgcatcgt ggtgtgctct gagaactctc tgcagttctt catgccagtg ctgggcgccc
     2821 tgttcatcgg agtggccgtg gcccctgcta acgacattta caacgagcgc gagctgctga
     2881 acagcatggg catttctcag cctaccgtgg tgttcgtgtc taagaagggc ctgcagaaga
     2941 tcctgaacgt gcagaagaag ctgcctatca tccagaagat catcatcatg gactctaaga
     3001 ccgactacca gggcttccag agcatgtaca cattcgtgac atctcatctg cctcctggct
     3061 tcaacgagta cgacttcgtg ccagagtctt tcgacaggga caaaaccatt gccctgatca
     3121 tgaacagctc tgggtctacc ggcctgccta agggcgtggc cctgcctcat cgcaccgcct
     3181 gtgtgcgctt ctctcacgcc cgcgacccta ttttcggcaa ccagatcatc cccgacaccg
     3241 ctattctgag cgtggtgcca ttccaccacg gcttcggcat gttcaccacc ctgggctacc
     3301 tgatttgcgg ctttcgggtg gtgctgatgt accgcttcga ggaggagctg ttcctgcgca
     3361 gcctgcaaga ctacaaaatt cagtctgccc tgctggtgcc aaccctgttc agcttcttcg
     3421 ctaagagcac cctgatcgac aagtacgacc tgtctaacct gcacgagatt gcctctggcg
     3481 gcgccccact gtctaaggag gtgggcgaag ccgtggccaa gcgctttcat ctgccaggca
     3541 tccgccaggg ctacggcctg accgagacaa ccagcgccat tctgattacc ccagagggcg
     3601 acgacaagcc tggcgccgtg ggcaaggtgg tgccattctt cgaggccaag gtggtggacc
     3661 tggacaccgg caagaccctg ggagtgaacc agcgcggcga gctgtgtgtg cgcggcccta
     3721 tgattatgtc cggctacgtg aataaccctg aggccacaaa cgccctgatc gacaaggacg
     3781 gctggctgca ctctggcgac attgcctact gggacgagga cgagcacttc ttcatcgtgg
     3841 accgcctgaa gtctctgatc aagtacaagg gctaccaggt ggccccagcc gagctggagt
     3901 ctatcctgct gcagcaccct aacattttcg acgccggagt ggccggcctg cccgacgacg
     3961 atgccggcga gctgcctgcc gccgtcgtcg tgctggaaca cggcaagacc atgaccgaga
     4021 aggagatcgt ggactatgtg gccagccagg tgacaaccgc caagaagctg cgcggcggag
     4081 tggtgttcgt ggacgaggtg cccaagggcc tgaccggcaa gctggacgcc cgcaagatcc
     4141 gcgagatcct gatcaaggct aagaaaggcg gcaagatcgc cgtgtaataa ttctagagtc
     4201 ggggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     4261 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     4321 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     4381 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     4441 gtaaaatcga taaggatcca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4501 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4561 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4621 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4681 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4741 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4801 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4861 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4921 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4981 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5041 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5101 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5161 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5221 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5281 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5341 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5401 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5461 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5521 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5581 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5641 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5701 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5761 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5821 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5881 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5941 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6001 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6061 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6121 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6181 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6241 tggccttttg ctggcctttt gctcacatgg ctcgac
//
