LOCUS       sequence-261903-        6269 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression vector, Luciferase reporter to test activity of
            miRNA (miR-144-3p).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6269)
  TITLE     Karreth lab plasmids
REFERENCE   2  (bases 1 to 6269)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: psiCHECK2-miR-144-3p
FEATURES             Location/Qualifiers
     source          1..6269
                     /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    1684..1732
                     /note="synthetic polyadenylation signal"
     promoter        1741..2492
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2522..2531
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2528..4180
                     /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             3487..3495
                     /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    4224..4345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4235..4254)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4289..4308
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4478..4582
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4583..4651,4652..5443)
                     /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(4801..4820)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5614..6202
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6103..6122
                     /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 cgctcgagag tacatcatct atactgtagc ggccgctggc
     1681 cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaggatcta
     1741 aatgagtctt cggacctcgc gggggccgct taagcggtgg ttagggtttg tctgacgcgg
     1801 ggggaggggg aaggaacgaa acactctcat tcggaggcgg ctcggggttt ggtcttggtg
     1861 gccacgggca cgcagaagag cgccgcgatc ctcttaagca cccccccgcc ctccgtggag
     1921 gcgggggttt ggtcggcggg tggtaactgg cgggccgctg actcgggcgg gtcgcgcgcc
     1981 ccagagtgtg accttttcgg tctgctcgca gacccccggg cggcgccgcc gcggcggcga
     2041 cgggctcgct gggtcctagg ctccatgggg accgtatacg tggacaggct ctggagcatc
     2101 cgcacgactg cggtgatatt accggagacc ttctgcggga cgagccgggt cacgcggctg
     2161 acgcggagcg tccgttgggc gacaaacacc aggacggggc acaggtacac tatcttgtca
     2221 cccggaggcg cgagggactg caggagcttc agggagtggc gcagctgctt catccccgtg
     2281 gcccgttgct cgcgtttgct ggcggtgtcc ccggaagaaa tatatttgca tgtctttagt
     2341 tctatgatga cacaaacccc gcccagcgtc ttgtcattgg cgaattcgaa cacgcagatg
     2401 cagtcggggc ggcgcggtcc caggtccact tcgcatatta aggtgacgcg tgtggcctcg
     2461 aacaccgagc gaccctgcag cgacccgctt aaaagcttgg cattccggta ctgttggtaa
     2521 agccaccatg gccgatgcta agaacattaa gaagggccct gctcccttct accctctgga
     2581 ggatggcacc gctggcgagc agctgcacaa ggccatgaag aggtatgccc tggtgcctgg
     2641 caccattgcc ttcaccgatg cccacattga ggtggacatc acctatgccg agtacttcga
     2701 gatgtctgtg cgcctggccg aggccatgaa gaggtacggc ctgaacacca accaccgcat
     2761 cgtggtgtgc tctgagaact ctctgcagtt cttcatgcca gtgctgggcg ccctgttcat
     2821 cggagtggcc gtggcccctg ctaacgacat ttacaacgag cgcgagctgc tgaacagcat
     2881 gggcatttct cagcctaccg tggtgttcgt gtctaagaag ggcctgcaga agatcctgaa
     2941 cgtgcagaag aagctgccta tcatccagaa gatcatcatc atggactcta agaccgacta
     3001 ccagggcttc cagagcatgt acacattcgt gacatctcat ctgcctcctg gcttcaacga
     3061 gtacgacttc gtgccagagt ctttcgacag ggacaaaacc attgccctga tcatgaacag
     3121 ctctgggtct accggcctgc ctaagggcgt ggccctgcct catcgcaccg cctgtgtgcg
     3181 cttctctcac gcccgcgacc ctattttcgg caaccagatc atccccgaca ccgctattct
     3241 gagcgtggtg ccattccacc acggcttcgg catgttcacc accctgggct acctgatttg
     3301 cggctttcgg gtggtgctga tgtaccgctt cgaggaggag ctgttcctgc gcagcctgca
     3361 agactacaaa attcagtctg ccctgctggt gccaaccctg ttcagcttct tcgctaagag
     3421 caccctgatc gacaagtacg acctgtctaa cctgcacgag attgcctctg gcggcgcccc
     3481 actgtctaag gaggtgggcg aagccgtggc caagcgcttt catctgccag gcatccgcca
     3541 gggctacggc ctgaccgaga caaccagcgc cattctgatt accccagagg gcgacgacaa
     3601 gcctggcgcc gtgggcaagg tggtgccatt cttcgaggcc aaggtggtgg acctggacac
     3661 cggcaagacc ctgggagtga accagcgcgg cgagctgtgt gtgcgcggcc ctatgattat
     3721 gtccggctac gtgaataacc ctgaggccac aaacgccctg atcgacaagg acggctggct
     3781 gcactctggc gacattgcct actgggacga ggacgagcac ttcttcatcg tggaccgcct
     3841 gaagtctctg atcaagtaca agggctacca ggtggcccca gccgagctgg agtctatcct
     3901 gctgcagcac cctaacattt tcgacgccgg agtggccggc ctgcccgacg acgatgccgg
     3961 cgagctgcct gccgccgtcg tcgtgctgga acacggcaag accatgaccg agaaggagat
     4021 cgtggactat gtggccagcc aggtgacaac cgccaagaag ctgcgcggcg gagtggtgtt
     4081 cgtggacgag gtgcccaagg gcctgaccgg caagctggac gcccgcaaga tccgcgagat
     4141 cctgatcaag gctaagaaag gcggcaagat cgccgtgtaa taattctaga gtcggggcgg
     4201 ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     4261 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     4321 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     4381 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     4441 cgataaggat ccaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4501 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4561 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4621 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4681 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4741 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4801 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4861 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4921 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4981 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5041 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5101 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     5161 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5221 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5281 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5341 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5401 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5461 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5521 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5581 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5641 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5701 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5761 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5821 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5881 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5941 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6001 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6061 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6121 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6181 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6241 ttgctggcct tttgctcaca tggctcgac
//
