LOCUS       sequence-322177-        6272 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector, Luciferase reporter to test activity of
            miRNA (miR-199a-5p).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6272)
  TITLE     Karreth lab plasmids
REFERENCE   2  (bases 1 to 6272)
  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-199a-5p
FEATURES             Location/Qualifiers
     source          1..6272
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        61..418
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        61..252
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(84..104)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      269..404
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          488..620
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     603..622
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        665..683
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      687..696
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             693..1628
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    1686..1734
                     /note="synthetic polyadenylation signal"
     promoter        1743..2494
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2524..2533
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2530..4182
                     /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             3489..3497
                     /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    4226..4347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4237..4256)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4291..4310
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4480..4584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4585..4653,4654..5445)
                     /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(4803..4822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5616..6204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6105..6124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatctgcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt
       61 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
      121 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
      181 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
      241 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
      301 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
      361 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
      421 gcttgattct tctgacacaa cagtctcgaa cttaagctgc agaagttggt cgtgaggcac
      481 tgggcaggta agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt
      541 gtcgagacag agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca
      601 ctttgccttt ctctccacag gtgtccactc ccagttcaat tacagctctt aaggctagag
      661 tacttaatac gactcactat aggctagcca ccatggcttc caaggtgtac gaccccgagc
      721 aacgcaaacg catgatcact gggcctcagt ggtgggctcg ctgcaagcaa atgaacgtgc
      781 tggactcctt catcaactac tatgattccg agaagcacgc cgagaacgcc gtgatttttc
      841 tgcatggtaa cgctgcctcc agctacctgt ggaggcacgt cgtgcctcac atcgagcccg
      901 tggctagatg catcatccct gatctgatcg gaatgggtaa gtccggcaag agcgggaatg
      961 gctcatatcg cctcctggat cactacaagt acctcaccgc ttggttcgag ctgctgaacc
     1021 ttccaaagaa aatcatcttt gtgggccacg actggggggc ttgtctggcc tttcactact
     1081 cctacgagca ccaagacaag atcaaggcca tcgtccatgc tgagagtgtc gtggacgtga
     1141 tcgagtcctg ggacgagtgg cctgacatcg aggaggatat cgccctgatc aagagcgaag
     1201 agggcgagaa aatggtgctt gagaataact tcttcgtcga gaccatgctc ccaagcaaga
     1261 tcatgcggaa actggagcct gaggagttcg ctgcctacct ggagccattc aaggagaagg
     1321 gcgaggttag acggcctacc ctctcctggc ctcgcgagat ccctctcgtt aagggaggca
     1381 agcccgacgt cgtccagatt gtccgcaact acaacgccta ccttcgggcc agcgacgatc
     1441 tgcctaagat gttcatcgag tccgaccctg ggttcttttc caacgctatt gtcgagggag
     1501 ctaagaagtt ccctaacacc gagttcgtga aggtgaaggg cctccacttc agccaggagg
     1561 acgctccaga tgaaatgggt aagtacatca agagcttcgt ggagcgcgtg ctgaagaacg
     1621 agcagtaatt ctaggcgatc gctcgaggaa caggtagtct gaacactggg gcggccgctg
     1681 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaggatc
     1741 taaatgagtc ttcggacctc gcgggggccg cttaagcggt ggttagggtt tgtctgacgc
     1801 ggggggaggg ggaaggaacg aaacactctc attcggaggc ggctcggggt ttggtcttgg
     1861 tggccacggg cacgcagaag agcgccgcga tcctcttaag cacccccccg ccctccgtgg
     1921 aggcgggggt ttggtcggcg ggtggtaact ggcgggccgc tgactcgggc gggtcgcgcg
     1981 ccccagagtg tgaccttttc ggtctgctcg cagacccccg ggcggcgccg ccgcggcggc
     2041 gacgggctcg ctgggtccta ggctccatgg ggaccgtata cgtggacagg ctctggagca
     2101 tccgcacgac tgcggtgata ttaccggaga ccttctgcgg gacgagccgg gtcacgcggc
     2161 tgacgcggag cgtccgttgg gcgacaaaca ccaggacggg gcacaggtac actatcttgt
     2221 cacccggagg cgcgagggac tgcaggagct tcagggagtg gcgcagctgc ttcatccccg
     2281 tggcccgttg ctcgcgtttg ctggcggtgt ccccggaaga aatatatttg catgtcttta
     2341 gttctatgat gacacaaacc ccgcccagcg tcttgtcatt ggcgaattcg aacacgcaga
     2401 tgcagtcggg gcggcgcggt cccaggtcca cttcgcatat taaggtgacg cgtgtggcct
     2461 cgaacaccga gcgaccctgc agcgacccgc ttaaaagctt ggcattccgg tactgttggt
     2521 aaagccacca tggccgatgc taagaacatt aagaagggcc ctgctccctt ctaccctctg
     2581 gaggatggca ccgctggcga gcagctgcac aaggccatga agaggtatgc cctggtgcct
     2641 ggcaccattg ccttcaccga tgcccacatt gaggtggaca tcacctatgc cgagtacttc
     2701 gagatgtctg tgcgcctggc cgaggccatg aagaggtacg gcctgaacac caaccaccgc
     2761 atcgtggtgt gctctgagaa ctctctgcag ttcttcatgc cagtgctggg cgccctgttc
     2821 atcggagtgg ccgtggcccc tgctaacgac atttacaacg agcgcgagct gctgaacagc
     2881 atgggcattt ctcagcctac cgtggtgttc gtgtctaaga agggcctgca gaagatcctg
     2941 aacgtgcaga agaagctgcc tatcatccag aagatcatca tcatggactc taagaccgac
     3001 taccagggct tccagagcat gtacacattc gtgacatctc atctgcctcc tggcttcaac
     3061 gagtacgact tcgtgccaga gtctttcgac agggacaaaa ccattgccct gatcatgaac
     3121 agctctgggt ctaccggcct gcctaagggc gtggccctgc ctcatcgcac cgcctgtgtg
     3181 cgcttctctc acgcccgcga ccctattttc ggcaaccaga tcatccccga caccgctatt
     3241 ctgagcgtgg tgccattcca ccacggcttc ggcatgttca ccaccctggg ctacctgatt
     3301 tgcggctttc gggtggtgct gatgtaccgc ttcgaggagg agctgttcct gcgcagcctg
     3361 caagactaca aaattcagtc tgccctgctg gtgccaaccc tgttcagctt cttcgctaag
     3421 agcaccctga tcgacaagta cgacctgtct aacctgcacg agattgcctc tggcggcgcc
     3481 ccactgtcta aggaggtggg cgaagccgtg gccaagcgct ttcatctgcc aggcatccgc
     3541 cagggctacg gcctgaccga gacaaccagc gccattctga ttaccccaga gggcgacgac
     3601 aagcctggcg ccgtgggcaa ggtggtgcca ttcttcgagg ccaaggtggt ggacctggac
     3661 accggcaaga ccctgggagt gaaccagcgc ggcgagctgt gtgtgcgcgg ccctatgatt
     3721 atgtccggct acgtgaataa ccctgaggcc acaaacgccc tgatcgacaa ggacggctgg
     3781 ctgcactctg gcgacattgc ctactgggac gaggacgagc acttcttcat cgtggaccgc
     3841 ctgaagtctc tgatcaagta caagggctac caggtggccc cagccgagct ggagtctatc
     3901 ctgctgcagc accctaacat tttcgacgcc ggagtggccg gcctgcccga cgacgatgcc
     3961 ggcgagctgc ctgccgccgt cgtcgtgctg gaacacggca agaccatgac cgagaaggag
     4021 atcgtggact atgtggccag ccaggtgaca accgccaaga agctgcgcgg cggagtggtg
     4081 ttcgtggacg aggtgcccaa gggcctgacc ggcaagctgg acgcccgcaa gatccgcgag
     4141 atcctgatca aggctaagaa aggcggcaag atcgccgtgt aataattcta gagtcggggc
     4201 ggccggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     4261 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     4321 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     4381 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa
     4441 atcgataagg atccaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4501 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4561 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4621 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4681 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4741 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4801 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4861 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4921 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4981 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5041 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5101 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5161 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5221 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5281 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5341 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5401 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5461 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5521 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5581 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5641 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5701 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5761 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5821 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5881 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5941 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6001 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6061 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6121 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6181 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6241 ttttgctggc cttttgctca catggctcga ca
//
