LOCUS       sequence-218577-        7856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Luciferase reporter containing PAK4 3' UTR with the second predicted
            MiR-199a-3p binding.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7856)
  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 7856)
  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: PAK4-3'UTR-BS2
FEATURES             Location/Qualifiers
     source          1..7856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        142..499
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        142..333
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(165..185)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      350..485
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      544..553
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             550..1482
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             1534..2325
                     /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(1585..1604)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2195..2214
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2389..2437
                     /note="synthetic polyadenylation signal"
     promoter        2593..2697
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2698..2766,2767..3558)
                     /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(2916..2935)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3716..4304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3796..3815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4420..4703
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     promoter        4760..5259
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4795..4818)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5192..5211
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      5300..5309
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5306..6958
                     /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    7632..7753
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7643..7662)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7697..7716
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg
       61 gtatttcaca ccgcatacgc ggatctgcgc agcaccatgg cctgaaataa cctctgaaag
      121 aggaacttgg ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
      181 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
      241 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
      301 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
      361 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
      421 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
      481 aggcctaggc ttttgcaaaa agcttgattc ttctgacaca acagtctcga accaaaggct
      541 ggagccacca tggcttccaa ggtgtacgac cccgagcaac gcaaacgcat gatcactggg
      601 cctcagtggt gggctcgctg caagcaaatg aacgtgctgg actccttcat caactactat
      661 gattccgaga agcacgccga gaacgccgtg atttttctgc atggtaacgc tgcctccagc
      721 tacctgtgga ggcacgtcgt gcctcacatc gagcccgtgg ctagatgcat catccctgat
      781 ctgatcggaa tgggtaagtc cggcaagagc gggaatggct catatcgcct cctggatcac
      841 tacaagtacc tcaccgcttg gttcgagctg ctgaaccttc caaagaaaat catctttgtg
      901 ggccacgact ggggggcttg tctggccttt cactactcct acgagcacca agacaagatc
      961 aaggccatcg tccatgctga gagtgtcgtg gacgtgatcg agtcctggga cgagtggcct
     1021 gacatcgagg aggatatcgc cctgatcaag agcgaagagg gcgagaaaat ggtgcttgag
     1081 aataacttct tcgtcgagac catgctccca agcaagatca tgcggaaact ggagcctgag
     1141 gagttcgctg cctacctgga gccattcaag gagaagggcg aggttagacg gcctaccctc
     1201 tcctggcctc gcgagatccc tctcgttaag ggaggcaagc ccgacgtcgt ccagattgtc
     1261 cgcaactaca acgcctacct tcgggccagc gacgatctgc ctaagatgtt catcgagtcc
     1321 gaccctgggt tcttttccaa cgctattgtc gagggagcta agaagttccc taacaccgag
     1381 ttcgtgaagg tgaagggcct ccacttcagc caggaggacg ctccagatga aatgggtaag
     1441 tacatcaaga gcttcgtgga gcgcgtgctg aagaacgagc agaccggtgg tgggagcgga
     1501 ggtggcggat caggtggcgg aggctccgga gggattgaac aagatggatt gcacgcaggt
     1561 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     1621 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     1681 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     1741 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     1801 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     1861 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     1921 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     1981 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     2041 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     2101 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     2161 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     2221 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     2281 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     2341 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg atggccgcaa taaaatatct
     2401 ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgatagcgat aaggatcctc
     2461 tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag
     2521 caccgtttct gcggactggc tttctacgta atggtttctt agacgtcagg tggcactttt
     2581 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2641 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2701 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2761 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     2821 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2881 gaacgttttc caatgatgag cactttcaaa gttctgctat gtggcgcggt attatcccgt
     2941 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3001 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3061 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aactatcgga
     3121 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3181 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3241 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3301 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3361 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3421 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3481 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3541 ctgattaagc attggtaatt cgaaatgacc gaccaagcga cgcccaaccg gtatcagctc
     3601 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3661 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3721 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3781 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3841 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3901 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3961 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4021 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4081 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4141 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4201 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4261 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatt tcaagaagat cctttgatct
     4321 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4381 gattatcaaa aaggatcttc acctagatcc ttttatagtc cggaaataca ggaacgcacg
     4441 ctggatggcc cttcgctggg atggtgaaac catgaaaaat ggcagcttca gtggattaag
     4501 tgggggtaat gtggcctgta ccctctggtt gcataggtat tcatacggtt aaaatttatc
     4561 aggcgcgatt gcggcagttt ttcgggtggt ttgttgccat ttttacctgt ctgctgccgt
     4621 gatcgcgctg aacgcgtttt agcggtgcgt acaattaagg gattatggta aatccactta
     4681 ctgtctgccc tcgtagccat cgagataaac cgcagtactc cggccacgat gcgtccggcg
     4741 tagaggatcg agatctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc
     4801 gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat
     4861 tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct
     4921 tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg
     4981 tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa
     5041 tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg
     5101 gctcagaggc tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg
     5161 gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct
     5221 gccgcgctgt tctcctcttc ctcatctccg ggcctttcga cctgcagccc aagcttggca
     5281 atccggtact gttggtaaag ccaccatgga agatgccaaa aacattaaga agggcccagc
     5341 gccattctac ccactcgaag acgggaccgc cggcgagcag ctgcacaaag ccatgaagcg
     5401 ctacgccctg gtgcccggca ccatcgcctt taccgacgca catatcgagg tggacattac
     5461 ctacgccgag tacttcgaga tgagcgttcg gctggcagaa gctatgaagc gctatgggct
     5521 gaatacaaac catcggatcg tggtgtgcag cgagaatagc ttgcagttct tcatgcccgt
     5581 gttgggtgcc ctgttcatcg gtgtggctgt ggccccagct aacgacatct acaacgagcg
     5641 cgagctgctg aacagcatgg gcatcagcca gcccaccgtc gtattcgtga gcaagaaagg
     5701 gctgcaaaag atcctcaacg tgcaaaagaa gctaccgatc atacaaaaga tcatcatcat
     5761 ggatagcaag accgactacc agggcttcca aagcatgtac accttcgtga cttcccattt
     5821 gccacccggc ttcaacgagt acgacttcgt gcccgagagc ttcgaccggg acaaaaccat
     5881 cgccctgatc atgaacagta gtggcagtac cggattgccc aagggcgtag ccctaccgca
     5941 ccgcaccgct tgtgtccgat tcagtcatgc ccgcgacccc atcttcggca accagatcat
     6001 ccccgacacc gctatcctca gcgtggtgcc atttcaccac ggcttcggca tgttcaccac
     6061 gctgggctac ttgatctgcg gctttcgggt cgtgctcatg taccgcttcg aggaggagct
     6121 attcttgcgc agcttgcaag actataagat tcaatctgcc ctgctggtgc ccacactatt
     6181 tagcttcttc gctaagagca ctctcatcga caagtacgac ctaagcaact tgcacgagat
     6241 cgccagcggc ggggcgccgc tcagcaagga ggtaggtgag gccgtggcca aacgcttcca
     6301 cctaccaggc atccgccagg gctacggcct gacagaaaca accagcgcca ttctgatcac
     6361 ccccgaaggg gacgacaagc ctggcgcagt aggcaaggtg gtgcccttct tcgaggctaa
     6421 ggtggtggac ttggacaccg gtaagacact gggtgtgaac cagcgcggcg agctgtgcgt
     6481 ccgtggcccc atgatcatga gcggctacgt taacaacccc gaggctacaa acgctctcat
     6541 cgacaaggac ggctggctgc acagcggcga catcgcctac tgggacgagg acgagcactt
     6601 cttcatcgtg gaccggctga agagcctgat caaatacaag ggctaccagg tagccccagc
     6661 cgaactggag agcatcctgc tgcaacaccc caacatcttc gacgccgggg tcgccggcct
     6721 gcccgacgac gatgccggcg agctgcccgc cgcagtcgtc gtgctggaac acggtaaaac
     6781 catgaccgag aaggagatcg tggactatgt ggccagccag gttacaaccg ccaagaagct
     6841 gcgcggtggt gttgtgttcg tggacgaggt gcctaaagga ctgaccggca agttggacgc
     6901 ccgcaagatc cgcgagattc tcattaaggc caagaagggc ggcaagatcg ccgtgtaatt
     6961 ctagttgttt aaacactgga agtctgcagt gggggtcgct gggggtggag agaacactaa
     7021 gaggtgaaca tgtatgagtg tgtgcacgcg tgtgagtgtg catgtgtgtg tgtgcaaagg
     7081 tccagccacc ccgtcctcca gcctgcaagg ggtgtctggc gccttgcctg acacccagcc
     7141 ccctctcccc ctgagccatt gtgggggtcg atcatgaatg tccgaagagt ggccttttcc
     7201 cgtagccctg cgcccccttt ctgtggctgg atggggagac aggtcagggc cccccaccct
     7261 ctccagcccc tgcagcaaat gactactgca cctggacagc ctcctctttt ctagaagtct
     7321 atttatattg tcattttata acactctagc ccctgccctt attgggggac agatggtccc
     7381 tgtcctgcgg ggtggccctg gcagaaccac tgcctgaaga accaggttcc tgcccggtca
     7441 gcgcagcccc agcccgccca cccctgcctc gagttagttt gagctcgcta gcctcgagtc
     7501 tagagtcgac ctgcaggcat gcaagctgat ccggctgcta acaaagcccg aaaggaagct
     7561 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ggccgcttcg
     7621 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     7681 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     7741 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat
     7801 gtgggaggtt tttttaagca agtaaaacct ctacaaatgt ggtaaaatcg aatttt
//
