LOCUS       sequence-216986-        7853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Luciferase reporter containing PAK4 3' UTR with the first predicted 
            MiR-199a-3p binding site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7853)
  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 7853)
  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-BS1
FEATURES             Location/Qualifiers
     source          1..7853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        99..456
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        99..290
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(122..142)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      307..442
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      501..510
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             507..1439
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             1491..2282
                     /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(1542..1561)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2152..2171
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2346..2394
                     /note="synthetic polyadenylation signal"
     promoter        2550..2654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2655..2723,2724..3515)
                     /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(2873..2892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3673..4261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3753..3772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4377..4660
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     promoter        4717..5216
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4752..4775)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5149..5168
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      5257..5266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5263..6915
                     /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    7586..7707
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7597..7616)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7651..7670
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 ccttacgcat ctgtgcggta tttcacaccg catacgcgga tctgcgcagc accatggcct
       61 gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg
      121 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
      181 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
      241 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
      301 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
      361 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
      421 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttgattcttc tgacacaaca
      481 gtctcgaacc aaaggctgga gccaccatgg cttccaaggt gtacgacccc gagcaacgca
      541 aacgcatgat cactgggcct cagtggtggg ctcgctgcaa gcaaatgaac gtgctggact
      601 ccttcatcaa ctactatgat tccgagaagc acgccgagaa cgccgtgatt tttctgcatg
      661 gtaacgctgc ctccagctac ctgtggaggc acgtcgtgcc tcacatcgag cccgtggcta
      721 gatgcatcat ccctgatctg atcggaatgg gtaagtccgg caagagcggg aatggctcat
      781 atcgcctcct ggatcactac aagtacctca ccgcttggtt cgagctgctg aaccttccaa
      841 agaaaatcat ctttgtgggc cacgactggg gggcttgtct ggcctttcac tactcctacg
      901 agcaccaaga caagatcaag gccatcgtcc atgctgagag tgtcgtggac gtgatcgagt
      961 cctgggacga gtggcctgac atcgaggagg atatcgccct gatcaagagc gaagagggcg
     1021 agaaaatggt gcttgagaat aacttcttcg tcgagaccat gctcccaagc aagatcatgc
     1081 ggaaactgga gcctgaggag ttcgctgcct acctggagcc attcaaggag aagggcgagg
     1141 ttagacggcc taccctctcc tggcctcgcg agatccctct cgttaaggga ggcaagcccg
     1201 acgtcgtcca gattgtccgc aactacaacg cctaccttcg ggccagcgac gatctgccta
     1261 agatgttcat cgagtccgac cctgggttct tttccaacgc tattgtcgag ggagctaaga
     1321 agttccctaa caccgagttc gtgaaggtga agggcctcca cttcagccag gaggacgctc
     1381 cagatgaaat gggtaagtac atcaagagct tcgtggagcg cgtgctgaag aacgagcaga
     1441 ccggtggtgg gagcggaggt ggcggatcag gtggcggagg ctccggaggg attgaacaag
     1501 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     1561 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     1621 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     1681 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     1741 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     1801 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     1861 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     1921 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     1981 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     2041 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     2101 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     2161 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     2221 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     2281 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgatg
     2341 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga
     2401 tagcgataag gatcctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga
     2461 cattcatccg gggtcagcac cgtttctgcg gactggcttt ctacgtaatg gtttcttaga
     2521 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2581 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2641 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2701 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2761 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2821 agagttttcg ccccgaagaa cgttttccaa tgatgagcac tttcaaagtt ctgctatgtg
     2881 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2941 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     3001 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3061 ttctgacaac tatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3121 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3181 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     3241 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3301 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3361 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3421 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3481 ctgagatagg tgcctcactg attaagcatt ggtaattcga aatgaccgac caagcgacgc
     3541 ccaaccggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3601 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3661 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3721 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3781 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3841 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3901 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3961 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4021 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4081 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     4141 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4201 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatttca
     4261 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4321 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt tatagtccgg
     4381 aaatacagga acgcacgctg gatggccctt cgctgggatg gtgaaaccat gaaaaatggc
     4441 agcttcagtg gattaagtgg gggtaatgtg gcctgtaccc tctggttgca taggtattca
     4501 tacggttaaa atttatcagg cgcgattgcg gcagtttttc gggtggtttg ttgccatttt
     4561 tacctgtctg ctgccgtgat cgcgctgaac gcgttttagc ggtgcgtaca attaagggat
     4621 tatggtaaat ccacttactg tctgccctcg tagccatcga gataaaccgc agtactccgg
     4681 ccacgatgcg tccggcgtag aggatcgaga tctaccgggt aggggaggcg cttttcccaa
     4741 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     4801 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg
     4861 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     4921 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     4981 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     5041 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     5101 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
     5161 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct
     5221 gcagcccaag cttggcaatc cggtactgtt ggtaaagcca ccatggaaga tgccaaaaac
     5281 attaagaagg gcccagcgcc attctaccca ctcgaagacg ggaccgccgg cgagcagctg
     5341 cacaaagcca tgaagcgcta cgccctggtg cccggcacca tcgcctttac cgacgcacat
     5401 atcgaggtgg acattaccta cgccgagtac ttcgagatga gcgttcggct ggcagaagct
     5461 atgaagcgct atgggctgaa tacaaaccat cggatcgtgg tgtgcagcga gaatagcttg
     5521 cagttcttca tgcccgtgtt gggtgccctg ttcatcggtg tggctgtggc cccagctaac
     5581 gacatctaca acgagcgcga gctgctgaac agcatgggca tcagccagcc caccgtcgta
     5641 ttcgtgagca agaaagggct gcaaaagatc ctcaacgtgc aaaagaagct accgatcata
     5701 caaaagatca tcatcatgga tagcaagacc gactaccagg gcttccaaag catgtacacc
     5761 ttcgtgactt cccatttgcc acccggcttc aacgagtacg acttcgtgcc cgagagcttc
     5821 gaccgggaca aaaccatcgc cctgatcatg aacagtagtg gcagtaccgg attgcccaag
     5881 ggcgtagccc taccgcaccg caccgcttgt gtccgattca gtcatgcccg cgaccccatc
     5941 ttcggcaacc agatcatccc cgacaccgct atcctcagcg tggtgccatt tcaccacggc
     6001 ttcggcatgt tcaccacgct gggctacttg atctgcggct ttcgggtcgt gctcatgtac
     6061 cgcttcgagg aggagctatt cttgcgcagc ttgcaagact ataagattca atctgccctg
     6121 ctggtgccca cactatttag cttcttcgct aagagcactc tcatcgacaa gtacgaccta
     6181 agcaacttgc acgagatcgc cagcggcggg gcgccgctca gcaaggaggt aggtgaggcc
     6241 gtggccaaac gcttccacct accaggcatc cgccagggct acggcctgac agaaacaacc
     6301 agcgccattc tgatcacccc cgaaggggac gacaagcctg gcgcagtagg caaggtggtg
     6361 cccttcttcg aggctaaggt ggtggacttg gacaccggta agacactggg tgtgaaccag
     6421 cgcggcgagc tgtgcgtccg tggccccatg atcatgagcg gctacgttaa caaccccgag
     6481 gctacaaacg ctctcatcga caaggacggc tggctgcaca gcggcgacat cgcctactgg
     6541 gacgaggacg agcacttctt catcgtggac cggctgaaga gcctgatcaa atacaagggc
     6601 taccaggtag ccccagccga actggagagc atcctgctgc aacaccccaa catcttcgac
     6661 gccggggtcg ccggcctgcc cgacgacgat gccggcgagc tgcccgccgc agtcgtcgtg
     6721 ctggaacacg gtaaaaccat gaccgagaag gagatcgtgg actatgtggc cagccaggtt
     6781 acaaccgcca agaagctgcg cggtggtgtt gtgttcgtgg acgaggtgcc taaaggactg
     6841 accggcaagt tggacgcccg caagatccgc gagattctca ttaaggccaa gaagggcggc
     6901 aagatcgccg tgtaattcta gttgtttaaa cccttcccct caaccaaaga gccccccggg
     6961 tcacccccgc cccactgagg ccagtagggg gccaggcctc ccactcctcc cagcccggga
     7021 gatgctccgc gtggcaccac cctccttgct gggggtagat gagaccctac tactgaactc
     7081 cagttttgat ctcgtgactt ttagaaaaac acagggactc gtgggagcaa gcgaggctcc
     7141 caggaccccc accctctggg acaggccctc ccccatgttc ttctgtctcc aggaagggca
     7201 gcggccctcc catcactgga agtctgcagt gggggtcgct gggggtggag agaacactaa
     7261 gaggtgaaca tgtatgagtg tgtgcacgcg tgtgagtgtg catgtgtgtg tgtgcaaagg
     7321 tccagccacc ccgtcctcca gcctgcaagg ggtgtctggc gccttgcctg acacccagcc
     7381 ccctctcccc ctgagccatt gtgggggtcg atcatgaatg tccgaagagt ggccgagctc
     7441 gctagcctcg agtctagagt cgacctgcag gcatgcaagc tgatccggct gctaacaaag
     7501 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     7561 gggcggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     7621 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     7681 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     7741 gttcaggggg agatgtggga ggttttttta agcaagtaaa acctctacaa atgtggtaaa
     7801 atcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tct
//
