LOCUS       sequence-322222-        6272 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression vector, Luciferase reporter to test activity of
            miRNA (miR-200b).
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 Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: psiCHECK2-miR-200b-3p
FEATURES             Location/Qualifiers
     source          1..6272
                     /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    1687..1735
                     /note="synthetic polyadenylation signal"
     promoter        1744..2495
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2525..2534
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2531..4183
                     /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             3490..3498
                     /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    4227..4348
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4238..4257)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4292..4311
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4481..4585
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4586..4654,4655..5446)
                     /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(4804..4823)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5617..6205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6106..6125
                     /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 cgctcgagaa actttattta tggcagtatt cgcggccgct
     1681 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaggat
     1741 ctaaatgagt cttcggacct cgcgggggcc gcttaagcgg tggttagggt ttgtctgacg
     1801 cggggggagg gggaaggaac gaaacactct cattcggagg cggctcgggg tttggtcttg
     1861 gtggccacgg gcacgcagaa gagcgccgcg atcctcttaa gcaccccccc gccctccgtg
     1921 gaggcggggg tttggtcggc gggtggtaac tggcgggccg ctgactcggg cgggtcgcgc
     1981 gccccagagt gtgacctttt cggtctgctc gcagaccccc gggcggcgcc gccgcggcgg
     2041 cgacgggctc gctgggtcct aggctccatg gggaccgtat acgtggacag gctctggagc
     2101 atccgcacga ctgcggtgat attaccggag accttctgcg ggacgagccg ggtcacgcgg
     2161 ctgacgcgga gcgtccgttg ggcgacaaac accaggacgg ggcacaggta cactatcttg
     2221 tcacccggag gcgcgaggga ctgcaggagc ttcagggagt ggcgcagctg cttcatcccc
     2281 gtggcccgtt gctcgcgttt gctggcggtg tccccggaag aaatatattt gcatgtcttt
     2341 agttctatga tgacacaaac cccgcccagc gtcttgtcat tggcgaattc gaacacgcag
     2401 atgcagtcgg ggcggcgcgg tcccaggtcc acttcgcata ttaaggtgac gcgtgtggcc
     2461 tcgaacaccg agcgaccctg cagcgacccg cttaaaagct tggcattccg gtactgttgg
     2521 taaagccacc atggccgatg ctaagaacat taagaagggc cctgctccct tctaccctct
     2581 ggaggatggc accgctggcg agcagctgca caaggccatg aagaggtatg ccctggtgcc
     2641 tggcaccatt gccttcaccg atgcccacat tgaggtggac atcacctatg ccgagtactt
     2701 cgagatgtct gtgcgcctgg ccgaggccat gaagaggtac ggcctgaaca ccaaccaccg
     2761 catcgtggtg tgctctgaga actctctgca gttcttcatg ccagtgctgg gcgccctgtt
     2821 catcggagtg gccgtggccc ctgctaacga catttacaac gagcgcgagc tgctgaacag
     2881 catgggcatt tctcagccta ccgtggtgtt cgtgtctaag aagggcctgc agaagatcct
     2941 gaacgtgcag aagaagctgc ctatcatcca gaagatcatc atcatggact ctaagaccga
     3001 ctaccagggc ttccagagca tgtacacatt cgtgacatct catctgcctc ctggcttcaa
     3061 cgagtacgac ttcgtgccag agtctttcga cagggacaaa accattgccc tgatcatgaa
     3121 cagctctggg tctaccggcc tgcctaaggg cgtggccctg cctcatcgca ccgcctgtgt
     3181 gcgcttctct cacgcccgcg accctatttt cggcaaccag atcatccccg acaccgctat
     3241 tctgagcgtg gtgccattcc accacggctt cggcatgttc accaccctgg gctacctgat
     3301 ttgcggcttt cgggtggtgc tgatgtaccg cttcgaggag gagctgttcc tgcgcagcct
     3361 gcaagactac aaaattcagt ctgccctgct ggtgccaacc ctgttcagct tcttcgctaa
     3421 gagcaccctg atcgacaagt acgacctgtc taacctgcac gagattgcct ctggcggcgc
     3481 cccactgtct aaggaggtgg gcgaagccgt ggccaagcgc tttcatctgc caggcatccg
     3541 ccagggctac ggcctgaccg agacaaccag cgccattctg attaccccag agggcgacga
     3601 caagcctggc gccgtgggca aggtggtgcc attcttcgag gccaaggtgg tggacctgga
     3661 caccggcaag accctgggag tgaaccagcg cggcgagctg tgtgtgcgcg gccctatgat
     3721 tatgtccggc tacgtgaata accctgaggc cacaaacgcc ctgatcgaca aggacggctg
     3781 gctgcactct ggcgacattg cctactggga cgaggacgag cacttcttca tcgtggaccg
     3841 cctgaagtct ctgatcaagt acaagggcta ccaggtggcc ccagccgagc tggagtctat
     3901 cctgctgcag caccctaaca ttttcgacgc cggagtggcc ggcctgcccg acgacgatgc
     3961 cggcgagctg cctgccgccg tcgtcgtgct ggaacacggc aagaccatga ccgagaagga
     4021 gatcgtggac tatgtggcca gccaggtgac aaccgccaag aagctgcgcg gcggagtggt
     4081 gttcgtggac gaggtgccca agggcctgac cggcaagctg gacgcccgca agatccgcga
     4141 gatcctgatc aaggctaaga aaggcggcaa gatcgccgtg taataattct agagtcgggg
     4201 cggccggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
     4261 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     4321 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     4381 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa
     4441 aatcgataag gatccaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     4501 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4561 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     4621 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     4681 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     4741 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     4801 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     4861 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     4921 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     4981 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5041 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5101 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5161 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5221 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5281 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5341 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5401 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     5461 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5521 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5581 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5641 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5701 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5761 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5821 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5881 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5941 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6001 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6061 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6121 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6181 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6241 cttttgctgg ccttttgctc acatggctcg ac
//
