LOCUS       sequence-322175-        6271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector, Luciferase reporter to test activity of
            miRNA (miR-223-3p).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6271)
  TITLE     Karreth lab plasmids
REFERENCE   2  (bases 1 to 6271)
  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-223-3p
FEATURES             Location/Qualifiers
     source          1..6271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        64..421
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        64..255
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(87..107)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      272..407
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          491..623
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     606..625
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        668..686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      690..699
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             696..1631
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    1688..1736
                     /note="synthetic polyadenylation signal"
     promoter        1745..2496
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2526..2535
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2532..4184
                     /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             3491..3499
                     /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    4228..4349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4239..4258)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4293..4312
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4482..4586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4587..4655,4656..5447)
                     /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(4805..4824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5618..6206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6107..6126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acagatctgc gcagcaccat ggcctgaaat aacctctgaa agaggaactt ggttaggtac
       61 cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
      121 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
      181 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      241 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
      301 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
      361 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
      421 aaagcttgat tcttctgaca caacagtctc gaacttaagc tgcagaagtt ggtcgtgagg
      481 cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg
      541 cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat
      601 ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct cttaaggcta
      661 gagtacttaa tacgactcac tataggctag ccaccatggc ttccaaggtg tacgaccccg
      721 agcaacgcaa acgcatgatc actgggcctc agtggtgggc tcgctgcaag caaatgaacg
      781 tgctggactc cttcatcaac tactatgatt ccgagaagca cgccgagaac gccgtgattt
      841 ttctgcatgg taacgctgcc tccagctacc tgtggaggca cgtcgtgcct cacatcgagc
      901 ccgtggctag atgcatcatc cctgatctga tcggaatggg taagtccggc aagagcggga
      961 atggctcata tcgcctcctg gatcactaca agtacctcac cgcttggttc gagctgctga
     1021 accttccaaa gaaaatcatc tttgtgggcc acgactgggg ggcttgtctg gcctttcact
     1081 actcctacga gcaccaagac aagatcaagg ccatcgtcca tgctgagagt gtcgtggacg
     1141 tgatcgagtc ctgggacgag tggcctgaca tcgaggagga tatcgccctg atcaagagcg
     1201 aagagggcga gaaaatggtg cttgagaata acttcttcgt cgagaccatg ctcccaagca
     1261 agatcatgcg gaaactggag cctgaggagt tcgctgccta cctggagcca ttcaaggaga
     1321 agggcgaggt tagacggcct accctctcct ggcctcgcga gatccctctc gttaagggag
     1381 gcaagcccga cgtcgtccag attgtccgca actacaacgc ctaccttcgg gccagcgacg
     1441 atctgcctaa gatgttcatc gagtccgacc ctgggttctt ttccaacgct attgtcgagg
     1501 gagctaagaa gttccctaac accgagttcg tgaaggtgaa gggcctccac ttcagccagg
     1561 aggacgctcc agatgaaatg ggtaagtaca tcaagagctt cgtggagcgc gtgctgaaga
     1621 acgagcagta attctaggcg atcgctcgag tggggtattt gacaaactga cagcggccgc
     1681 tggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgagga
     1741 tctaaatgag tcttcggacc tcgcgggggc cgcttaagcg gtggttaggg tttgtctgac
     1801 gcggggggag ggggaaggaa cgaaacactc tcattcggag gcggctcggg gtttggtctt
     1861 ggtggccacg ggcacgcaga agagcgccgc gatcctctta agcacccccc cgccctccgt
     1921 ggaggcgggg gtttggtcgg cgggtggtaa ctggcgggcc gctgactcgg gcgggtcgcg
     1981 cgccccagag tgtgaccttt tcggtctgct cgcagacccc cgggcggcgc cgccgcggcg
     2041 gcgacgggct cgctgggtcc taggctctat ggggaccgta tacgtggaca ggctctggag
     2101 catccgcacg actgcggtga tattaccgga gaccttctgc gggacgagcc gggtcacgcg
     2161 gctgacgcgg agcgtccgtt gggcgacaaa caccaggacg gggcacaggt acactatctt
     2221 gtcacccgga ggcgcgaggg actgcaggag cttcagggag tggcgcagct gcttcatccc
     2281 cgtggcccgt tgctcgcgtt tgctggcggt gtccccggaa gaaatatatt tgcatgtctt
     2341 tagttctatg atgacacaaa ccccgcccag cgtcttgtca ttggcgaatt cgaacacgca
     2401 gatgcagtcg gggcggcgcg gtcccaggtc cacttcgcat attaaggtga cgcgtgtggc
     2461 ctcgaacacc gagcgaccct gcagcgaccc gcttaaaagc ttggcattcc ggtactgttg
     2521 gtaaagccac catggccgat gctaagaaca ttaagaaggg ccctgctccc ttctaccctc
     2581 tggaggatgg caccgctggc gagcagctgc acaaggccat gaagaggtat gccctggtgc
     2641 ctggcaccat tgccttcacc gatgcccaca ttgaggtgga catcacctat gccgagtact
     2701 tcgagatgtc tgtgcgcctg gccgaggcca tgaagaggta cggcctgaac accaaccacc
     2761 gcatcgtggt gtgctctgag aactctctgc agttcttcat gccagtgctg ggcgccctgt
     2821 tcatcggagt ggccgtggcc cctgctaacg acatttacaa cgagcgcgag ctgctgaaca
     2881 gcatgggcat ttctcagcct accgtggtgt tcgtgtctaa gaagggcctg cagaagatcc
     2941 tgaacgtgca gaagaagctg cctatcatcc agaagatcat catcatggac tctaagaccg
     3001 actaccaggg cttccagagc atgtacacat tcgtgacatc tcatctgcct cctggcttca
     3061 acgagtacga cttcgtgcca gagtctttcg acagggacaa aaccattgcc ctgatcatga
     3121 acagctctgg gtctaccggc ctgcctaagg gcgtggccct gcctcatcgc accgcctgtg
     3181 tgcgcttctc tcacgcccgc gaccctattt tcggcaacca gatcatcccc gacaccgcta
     3241 ttctgagcgt ggtgccattc caccacggct tcggcatgtt caccaccctg ggctacctga
     3301 tttgcggctt tcgggtggtg ctgatgtacc gcttcgagga ggagctgttc ctgcgcagcc
     3361 tgcaagacta caaaattcag tctgccctgc tggtgccaac cctgttcagc ttcttcgcta
     3421 agagcaccct gatcgacaag tacgacctgt ctaacctgca cgagattgcc tctggcggcg
     3481 ccccactgtc taaggaggtg ggcgaagccg tggccaagcg ctttcatctg ccaggcatcc
     3541 gccagggcta cggcctgacc gagacaacca gcgccattct gattacccca gagggcgacg
     3601 acaagcctgg cgccgtgggc aaggtggtgc cattcttcga ggccaaggtg gtggacctgg
     3661 acaccggcaa gaccctggga gtgaaccagc gcggcgagct gtgtgtgcgc ggccctatga
     3721 ttatgtccgg ctacgtgaat aaccctgagg ccacaaacgc cctgatcgac aaggacggct
     3781 ggctgcactc tggcgacatt gcctactggg acgaggacga gcacttcttc atcgtggacc
     3841 gcctgaagtc tctgatcaag tacaagggct accaggtggc cccagccgag ctggagtcta
     3901 tcctgctgca gcaccctaac attttcgacg ccggagtggc cggcctgccc gacgacgatg
     3961 ccggcgagct gcctgccgcc gtcgtcgtgc tggaacacgg caagaccatg accgagaagg
     4021 agatcgtgga ctatgtggcc agccaggtga caaccgccaa gaagctgcgc ggcggagtgg
     4081 tgttcgtgga cgaggtgccc aagggcctga ccggcaagct ggacgcccgc aagatccgcg
     4141 agatcctgat caaggctaag aaaggcggca agatcgccgt gtaataattc tagagtcggg
     4201 gcggccggcc gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa
     4261 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4321 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     4381 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     4441 aaatcgataa ggatccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4501 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4561 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     4621 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     4681 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     4741 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     4801 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     4861 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     4921 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     4981 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5041 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5101 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5161 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5221 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5281 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5341 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5401 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5461 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5521 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5581 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5641 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5701 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5761 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5821 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5881 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5941 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6001 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6061 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6121 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6181 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6241 ccttttgctg gccttttgct cacatggctc g
//
