LOCUS       sequence-304706-        8982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Luciferase reporter assay for microRNA binding on the RECK 3'UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8982)
  AUTHORS   Jung HM, Phillips BL, Patel RS, Cohen DM, Jakymiw A, Kong WW, Cheng 
            JQ, Chan EK
  TITLE     Keratinization-associated miR-7 and miR-21 regulate tumor suppressor
            reversion-inducing cysteine-rich protein with kazal motifs (RECK) in
            oral cancer.
  JOURNAL   J Biol Chem. 2012 Aug 24;287(35):29261-72. doi: 
            10.1074/jbc.M112.366518. Epub 2012 Jul 2.
  PUBMED    22761427
REFERENCE   2  (bases 1 to 8982)
  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: 
            "10.1074/jbc.M112.366518"; journalName: "J Biol Chem"; date: 
            "2012-08-24- 24"; volume: "287"; issue: "35"; pages: "29261-72"
COMMENT     Sequence Label: pMiR-RECK-3'UTR
FEATURES             Location/Qualifiers
     source          1..8982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..795
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGMADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(55..74)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     665..684
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    816..1382
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    832..1384
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(976..993)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1203..1222
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(4493..4512)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4537..4584
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4913..5501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5402..5421
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5650..6272
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     6054..6073
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     6283..6299
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6283..6299
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(6319..6342)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(6361..6390)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(6402..7094)
                     /codon_start=1
                     /product="red fluorescent protein from Entacmaea 
                     quadricolor"
                     /label=TurboRFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     primer_bind     complement(7150..7169)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7151..7169)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(7195..7398)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(7224..7244)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        7399..7778
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(7939..7956)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7939..7955)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7948..7970)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(8096..8551)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8233..8254)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8445..8464
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        8578..8682
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8707..8727)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      8800..8934
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
       61 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
      121 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
      181 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
      241 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
      301 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
      361 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
      421 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
      481 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
      541 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
      601 ggccgctttt ctggattcat cgactgtggc cggctgggta tggcggatcg ctatcaggac
      661 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
      721 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
      781 gacgagttct tctgagcggg actctggggt tcgattccgc ccccccccct aacgttactg
      841 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
      901 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
      961 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     1021 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     1081 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     1141 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     1201 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     1261 gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa
     1321 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat
     1381 aatatggcca caacaattga tggaagacgc caaaaacata aagaaaggcc cagcgccatt
     1441 ctacccactc gaagacggga ccgccggcga gcagctgcac aaagccatga agcgctacgc
     1501 cctggtgccc ggcaccatcg cctttaccga cgcacatatc gaggtggaca ttacctacgc
     1561 cgagtacttc gagatgagcg ttcggctggc agaagctatg aagcgctatg ggctgaatac
     1621 aaaccatcgg atcgtggtgt gcagcgagaa tagcttgcag ttcttcatgc ccgtgttggg
     1681 tgccctgttc atcggtgtgg ctgtggcccc agctaacgac atctacaacg agcgcgagct
     1741 gctgaacagc atgggcatca gccagcccac cgtcgtattc gtgagcaaga aagggctgca
     1801 aaagatcctc aacgtgcaaa agaagctacc gatcatacaa aagatcatca tcatggatag
     1861 caagaccgac taccagggct tccaaagcat gtacaccttc gtgacttccc atttgccacc
     1921 cggcttcaac gagtacgact tcgtgcccga gagcttcgac cgggacaaaa ccatcgccct
     1981 gatcatgaac agtagtggca gtaccggatt gcccaagggc gtagccctac cgcaccgcac
     2041 cgcttgtgtc cgattcagtc atgcccgcga ccccatcttc ggcaaccaga tcatccccga
     2101 caccgctatc ctcagcgtgg tgccatttca ccacggcttc ggcatgttca ccacgctggg
     2161 ctacttgatc tgcggctttc gggtcgtgct catgtaccgc ttcgaggagg agctattctt
     2221 gcgcagcttg caagactata agattcaatc tgccctgctg gtgcccacac tatttagctt
     2281 cttcgctaag agcactctca tcgacaagta cgacctaagc aacttgcacg agatcgccag
     2341 cggcggggcg ccgctcagca aggaggtagg tgaggccgtg gccaaacgct tccacctacc
     2401 aggcatccgc cagggctacg gcctgacaga aacaaccagc gccattctga tcacccccga
     2461 aggggacgac aagcctggcg cagtaggcaa ggtggtgccc ttcttcgagg ctaaggtggt
     2521 ggacttggac accggtaaga cactgggtgt gaaccagcgc ggcgagctgt gcgtccgtgg
     2581 ccccatgatc atgagcggct acgttaacaa ccccgaggct acaaacgctc tcatcgacaa
     2641 ggacggctgg ctgcacagcg gcgacatcgc ctactgggac gaggacgagc acttcttcat
     2701 cgtggaccgg ctgaagagcc tgatcaaata caagggctac caggtagccc cagccgaact
     2761 ggagagcatc ctgctgcaac accccaacat cttcgacgcc ggggtcgccg gcctgcccga
     2821 cgacgatgcc ggcgagctgc ccgccgcagt cgtcgtgctg gaacacggta aaaccatgac
     2881 cgagaaggag atcgtggact atgtggccag ccaggttaca accgccaaga agctgcgcgg
     2941 tggtgttgtg ttcgtggacg aggtgcctaa aggactgacc ggcaagttgg acgcccgcaa
     3001 gatccgcgag attctcatta aggccaagaa gggcggaaag atcgccgtgt aacaattggc
     3061 agagctcaga attcaggact gctggtttgt agttgaatat tggccaagga aaggcacatg
     3121 tcacctctat tcgccacaca gtattttttt ttttaatccg ccaatattag taggattttt
     3181 gttttgtttt tacaaatgtt aaaatgtgtt gttccaaata ctaatgaaaa cagaatgtct
     3241 cttcctggta gaccactgcc atatgattta catttcctca ccataagggt cccccactct
     3301 aaagcaaatt tatcgctggg aaatgagatg accacttttt agaaagataa ttcactgtac
     3361 tatcaggttc acaaacttca tttcagagtt ctttttgaag tatttaaggt tcccgttgca
     3421 tttgttttgt ttacagataa ttacctactc tggctagaag ctaggggtcc cagtgaagag
     3481 ccactgccat taaagaatat gaaacataga taaaacatct ttgaaattat gtaaattatg
     3541 taaattatca ggcaaatttg cattaaatta cagaaattta attcagaacc ccaactactg
     3601 tgttatgcaa aagcaagctg attaaatgac actcatataa ttatatgttg taagcaacag
     3661 gctcactggt cacggatttg tgtctgtgac ttttgtgaaa gggagaagtg acattgcatc
     3721 aaagcatctt gcattatgca atttttatat taaccagata tatattcatc ggtattcatc
     3781 caagttaaat gtagagtttt taaacatcaa tctttaaacc aattgctgct acttatataa
     3841 ttgccaaaaa gtgaaataat gtgtagttca tgtaaataat acattatatt tctattttat
     3901 tatgaagaag gtgaatagcc atatttgtaa aatgacaatc atgtgtgtta acccagtgct
     3961 ttccattcgt gaaaacacat ttgctttttg tgatatgcac aatgtagata agtgttctgt
     4021 ctgactttct tttttgatat agaagtataa agaattgtgg tttatatatt taaaagtgtc
     4081 aagctgagta ttaaaatgta tgcatgttgt ctaagaaatt gaatactttg aatgtgtttc
     4141 acagtttgaa ataagctatt tgatgtaata cttcttgtgt gtatgcacat gaacttagat
     4201 tttacatgaa gtattttttc agtattatat gtaccctctg aaatacatag ggatatgcgt
     4261 attataccaa aatgttgctg aaaaatgggc acttaaagcg gccgcggcat ctagattcga
     4321 agaaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4381 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4441 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     4501 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     4561 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     4621 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     4681 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     4741 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     4801 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4861 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4921 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4981 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5041 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5101 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5161 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5221 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5281 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5341 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5401 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5461 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5521 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5581 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgctggc
     5641 cgcgtaccca attcaacagg catctactga gtggacccaa cgcatgagag gacagtgcca
     5701 agcaagcaac tcaaatgtcc caccggttgg gcatggccag gtagcctatg ctgtgtctgg
     5761 acgtcctcct gctggtatag ttattttaaa atcagaagga cagggaaggg agcagtggtt
     5821 cacgcctgta atcccagcaa tttgggaggc caaggtgggt agatcacctg agattaggag
     5881 ttggagacca gcctggccaa tatggtgaaa ccccgtctct accaaaaaaa caaaaattag
     5941 ctgagcctgg tcatgcatgc ctggaatccc aacaactcgg gaggctgagg caggagaatc
     6001 gcttgaaccc aggaggcgga gattgcagtg agccaagatt gtgccactgc actccagctt
     6061 ggttcccaat agaccccgca ggccctacag gttgtcttcc caacttgccc cttgctccat
     6121 accacccccc tccaccccat aatattatag aaggacacct agtcagacaa aatgatgcaa
     6181 cttaatttta ttaggacaag gctggtgggc actggagtgg caacttccag ggccaggaga
     6241 ggcactgggg aggggtcaca gggatgccac ccgggatctg ttcaggaaac agctatgacc
     6301 gcggccggcc gtttaaacct tatcgtcgtc atccttgtaa tccaggatat catttgctgc
     6361 cagatcctct tctgagatga gtttctgctc gagggacgcg atctgtgccc cagtttgcta
     6421 gggaggtcgc agtacttggc cacagccatc tcgtgctgct cgacgtaggt ctctttgtcg
     6481 gcctccttga ttctttccag tctgtggtcc acgaagtgga agccgggcat cttgaggttc
     6541 ttagcgggtt tcttggatct gtatgtggtc ttgaaggagc agtgcaggta gcccccgccc
     6601 acgagcttca gggccatctg gctgtggcct ctcaggccgc cgtcagcggg gtacagcatc
     6661 tcggtgttgg cctcccagcc gcgtgttttc ttctgcatca cagggccgtt ggatgggaag
     6721 ttcaccccgt tgatcttgac gttgtagatg atgcagccgt tctggaagct ggtgtcctgg
     6781 gtagcggtca gcacgccccc gtcttcgtat gtggtgattc tctcccatgt gaagccctca
     6841 gggaaggact gcttaaagaa gtcggggatg ccctgggtgt ggttgatgaa ggctttgctg
     6901 ccgtacatga agctggtagc caggatgtcg aaggcgaagg ggagagggcc gccctcgacc
     6961 accttgatct tcatggtctg ggtgccctcg tagggcttgc cttcgccctc ggatgtgcac
     7021 ttgaagtggt ggttgttcac ggtgccctcc atgtacagct tcatgtgcat gttctccttg
     7081 atcagctcgc tcatggcgaa cgcggcgcag atctcctcgg tactggatcc agtcgacgat
     7141 tcccggccgc cctatagtga gtcgtattac aaaattctga cggttcacta aacgagctct
     7201 gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaacg gggcggggtt
     7261 attacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc
     7321 aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattggtgta
     7381 ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt
     7441 aggaaagtcc cgtaaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt
     7501 gacgtcaata gggggcggac ttggcatatg atacacttga tgtactgcca agtgggcagt
     7561 ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg
     7621 aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc
     7681 atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt
     7741 aattgattac tattaataac tagtcaataa tcaatgtcaa catggcggtc atattggaca
     7801 tgagccaata taaatgtaca tattatgata tagatacaac gtatgcaatg gccaatagcc
     7861 aatattgatt tatgctatat aaccaatgac taatatggct aattgccaat attgattcaa
     7921 tgtatagatc agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     7981 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     8041 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt
     8101 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     8161 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     8221 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     8281 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     8341 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     8401 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     8461 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     8521 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     8581 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     8641 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     8701 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     8761 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     8821 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     8881 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     8941 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gc
//
