LOCUS       sequence-216988-        8139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Luciferase reporter containing PAK4-FL- 3' UTR with both the 
            predicted MiR-199a-3p binding sites mutated.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8139)
  AUTHORS   Phatak P, Burrows WM, Chesnick IE, Tulapurkar ME, Rao JN, Turner DJ,
            Hamburger AW, Wang JY, Donahue JM
  TITLE     MiR-199a-3p decreases esophageal cancer cell proliferation by 
            targeting p21 activated kinase 4.
  JOURNAL   Oncotarget. 2018 Jun 19;9(47):28391-28407. doi: 
            10.18632/oncotarget.25375. eCollection 2018 Jun 19.
  PUBMED    29983868
REFERENCE   2  (bases 1 to 8139)
  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"; doi: 
            "10.18632/oncotarget.25375"; journalName: "Oncotarget"; date: 
            "2018-06-19- 19"; volume: "9"; issue: "47"; pages: "28391-28407"
COMMENT     Sequence Label: PAK4-FL-3'UTR-MT-both
FEATURES             Location/Qualifiers
     source          1..8139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..41)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     415..434
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      523..532
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             529..2181
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    3138..3259
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3149..3168)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3203..3222
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3504..3861
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3504..3695
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3527..3547)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3712..3847
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      3906..3915
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3912..4844
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             4896..5687
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4947..4966)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5557..5576
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5751..5799
                     /note="synthetic polyadenylation signal"
     promoter        5955..6059
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6060..6128,6129..6920)
                     /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(6278..6297)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(7078..7666)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7158..7177)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7782..8065
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     promoter        join(8122..8139,1..482)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
ORIGIN
        1 tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa
       61 gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct
      121 ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc
      181 cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc
      241 agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc
      301 agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
      361 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
      421 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
      481 cgacctgcag cccaagcttg gcaatccggt actgttggta aagccaccat ggaagatgcc
      541 aaaaacatta agaagggccc agcgccattc tacccactcg aagacgggac cgccggcgag
      601 cagctgcaca aagccatgaa gcgctacgcc ctggtgcccg gcaccatcgc ctttaccgac
      661 gcacatatcg aggtggacat tacctacgcc gagtacttcg agatgagcgt tcggctggca
      721 gaagctatga agcgctatgg gctgaataca aaccatcgga tcgtggtgtg cagcgagaat
      781 agcttgcagt tcttcatgcc cgtgttgggt gccctgttca tcggtgtggc tgtggcccca
      841 gctaacgaca tctacaacga gcgcgagctg ctgaacagca tgggcatcag ccagcccacc
      901 gtcgtattcg tgagcaagaa agggctgcaa aagatcctca acgtgcaaaa gaagctaccg
      961 atcatacaaa agatcatcat catggatagc aagaccgact accagggctt ccaaagcatg
     1021 tacaccttcg tgacttccca tttgccaccc ggcttcaacg agtacgactt cgtgcccgag
     1081 agcttcgacc gggacaaaac catcgccctg atcatgaaca gtagtggcag taccggattg
     1141 cccaagggcg tagccctacc gcaccgcacc gcttgtgtcc gattcagtca tgcccgcgac
     1201 cccatcttcg gcaaccagat catccccgac accgctatcc tcagcgtggt gccatttcac
     1261 cacggcttcg gcatgttcac cacgctgggc tacttgatct gcggctttcg ggtcgtgctc
     1321 atgtaccgct tcgaggagga gctattcttg cgcagcttgc aagactataa gattcaatct
     1381 gccctgctgg tgcccacact atttagcttc ttcgctaaga gcactctcat cgacaagtac
     1441 gacctaagca acttgcacga gatcgccagc ggcggggcgc cgctcagcaa ggaggtaggt
     1501 gaggccgtgg ccaaacgctt ccacctacca ggcatccgcc agggctacgg cctgacagaa
     1561 acaaccagcg ccattctgat cacccccgaa ggggacgaca agcctggcgc agtaggcaag
     1621 gtggtgccct tcttcgaggc taaggtggtg gacttggaca ccggtaagac actgggtgtg
     1681 aaccagcgcg gcgagctgtg cgtccgtggc cccatgatca tgagcggcta cgttaacaac
     1741 cccgaggcta caaacgctct catcgacaag gacggctggc tgcacagcgg cgacatcgcc
     1801 tactgggacg aggacgagca cttcttcatc gtggaccggc tgaagagcct gatcaaatac
     1861 aagggctacc aggtagcccc agccgaactg gagagcatcc tgctgcaaca ccccaacatc
     1921 ttcgacgccg gggtcgccgg cctgcccgac gacgatgccg gcgagctgcc cgccgcagtc
     1981 gtcgtgctgg aacacggtaa aaccatgacc gagaaggaga tcgtggacta tgtggccagc
     2041 caggttacaa ccgccaagaa gctgcgcggt ggtgttgtgt tcgtggacga ggtgcctaaa
     2101 ggactgaccg gcaagttgga cgcccgcaag atccgcgaga ttctcattaa ggccaagaag
     2161 ggcggcaaga tcgccgtgta attctagttg tttaaaccct tcccctcaac caaagagccc
     2221 cccgggtcac ccccgcccca ctgaggccag tagggggcca ggcctcccac tcctcccagc
     2281 ccgggagatg ctccgtgtgg caccaccctc cttgctgggg gtagatgaga ccctactgac
     2341 aaactccagt tttgatctcg tgacttttag aaaaacacag ggactcgtgg gagcaagcga
     2401 ggctcccagg acccccaccc tctgggacag gccctccccc atgttcttct gtctccagga
     2461 agggcagcgg ccctcccatc actggaagtc tgcagtgggg gtcgctgggg gtggagagaa
     2521 cactaagagg tgaacatgta tgagtgtgtg cacgcgtgtg agtgtgcatg tgtgtgtgtg
     2581 caaaggtcca gccaccccgt cctccagcct gcaaggggtg tctggcgcct tgcctgacac
     2641 ccagccccct ctccccctga gccattgtgg gggtcgatca tgaatgtccg aagagtggcc
     2701 ttttcccgta gccctgcgcc ccctttctgt ggctggatgg ggagacaggt cagggccccc
     2761 caccctctcc agcccctgca gcaaatgact gacacacctg gacagcctcc tcttttctag
     2821 aagtctattt atattgtcat tttataacac tctagcccct gcccttattg ggggacagat
     2881 ggtccctgtc ctgcggggtg gccctggcag aaccactgcc tgaagaacca ggttcctgcc
     2941 cggtcagcgc agccccagcc cgcccacccc tgcctcgagt tagtttgagc tcgctagcct
     3001 cgagtctaga gtcgacctgc aggcatgcaa gctgatccgg ctgctaacaa agcccgaaag
     3061 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcggcc
     3121 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     3181 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     3241 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     3301 ggagatgtgg gaggtttttt taagcaagta aaacctctac aaatgtggta aaatcgaatt
     3361 ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     3421 cggtatttca caccgcatac gcggatctgc gcagcaccat ggcctgaaat aacctctgaa
     3481 agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3541 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3601 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3661 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3721 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3781 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3841 ggaggcctag gcttttgcaa aaagcttgat tcttctgaca caacagtctc gaaccaaagg
     3901 ctggagccac catggcttcc aaggtgtacg accccgagca acgcaaacgc atgatcactg
     3961 ggcctcagtg gtgggctcgc tgcaagcaaa tgaacgtgct ggactccttc atcaactact
     4021 atgattccga gaagcacgcc gagaacgccg tgatttttct gcatggtaac gctgcctcca
     4081 gctacctgtg gaggcacgtc gtgcctcaca tcgagcccgt ggctagatgc atcatccctg
     4141 atctgatcgg aatgggtaag tccggcaaga gcgggaatgg ctcatatcgc ctcctggatc
     4201 actacaagta cctcaccgct tggttcgagc tgctgaacct tccaaagaaa atcatctttg
     4261 tgggccacga ctggggggct tgtctggcct ttcactactc ctacgagcac caagacaaga
     4321 tcaaggccat cgtccatgct gagagtgtcg tggacgtgat cgagtcctgg gacgagtggc
     4381 ctgacatcga ggaggatatc gccctgatca agagcgaaga gggcgagaaa atggtgcttg
     4441 agaataactt cttcgtcgag accatgctcc caagcaagat catgcggaaa ctggagcctg
     4501 aggagttcgc tgcctacctg gagccattca aggagaaggg cgaggttaga cggcctaccc
     4561 tctcctggcc tcgcgagatc cctctcgtta agggaggcaa gcccgacgtc gtccagattg
     4621 tccgcaacta caacgcctac cttcgggcca gcgacgatct gcctaagatg ttcatcgagt
     4681 ccgaccctgg gttcttttcc aacgctattg tcgagggagc taagaagttc cctaacaccg
     4741 agttcgtgaa ggtgaagggc ctccacttca gccaggagga cgctccagat gaaatgggta
     4801 agtacatcaa gagcttcgtg gagcgcgtgc tgaagaacga gcagaccggt ggtgggagcg
     4861 gaggtggcgg atcaggtggc ggaggctccg gagggattga acaagatgga ttgcacgcag
     4921 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4981 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     5041 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     5101 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     5161 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     5221 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     5281 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     5341 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     5401 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     5461 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     5521 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     5581 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     5641 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     5701 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgatggccgc aataaaatat
     5761 ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagcg ataaggatcc
     5821 tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc
     5881 agcaccgttt ctgcggactg gctttctacg taatggtttc ttagacgtca ggtggcactt
     5941 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     6001 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     6061 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     6121 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     6181 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     6241 aagaacgttt tccaatgatg agcactttca aagttctgct atgtggcgcg gtattatccc
     6301 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     6361 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     6421 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaactatcg
     6481 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6541 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6601 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6661 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6721 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6781 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6841 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6901 cactgattaa gcattggtaa ttcgaaatga ccgaccaagc gacgcccaac cggtatcagc
     6961 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     7021 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     7081 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     7141 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     7201 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     7261 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     7321 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     7381 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     7441 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     7501 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     7561 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     7621 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tttcaagaag atcctttgat
     7681 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7741 gagattatca aaaaggatct tcacctagat ccttttatag tccggaaata caggaacgca
     7801 cgctggatgg cccttcgctg ggatggtgaa accatgaaaa atggcagctt cagtggatta
     7861 agtgggggta atgtggcctg taccctctgg ttgcataggt attcatacgg ttaaaattta
     7921 tcaggcgcga ttgcggcagt ttttcgggtg gtttgttgcc atttttacct gtctgctgcc
     7981 gtgatcgcgc tgaacgcgtt ttagcggtgc gtacaattaa gggattatgg taaatccact
     8041 tactgtctgc cctcgtagcc atcgagataa accgcagtac tccggccacg atgcgtccgg
     8101 cgtagaggat cgagatctac cgggtagggg aggcgcttt
//
