LOCUS       sequence-396464-        4404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid with shRNAmiR targeting RKIP (PEBP1), with a BFP 
            reporter on the backbone. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4404)
  AUTHORS   Wan Y, Cohen J, Szenk M, Farquhar KS, Coraci D, Krzyszton R, Azukas 
            J, Van Nest N, Smashnov A, Chern YJ, De Martino D, Nguyen LC, Bien 
            H, Bravo-Cordero JJ, Chan CH, Rosner MR, Balazsi G
  TITLE     Nonmonotone invasion landscape by noise-aware control of metastasis 
            activator levels.
  JOURNAL   Nat Chem Biol. 2023 Jul;19(7):887-899. doi: 
            10.1038/s41589-023-01344-z. Epub 2023 May 25.
  PUBMED    37231268
REFERENCE   2  (bases 1 to 4404)
  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: 
            "10.1038/s41589-023-01344-z"; journalName: "Nat Chem Biol"; date: 
            "2023-07"; volume: "19"; issue: "7"; pages: "887-899"
COMMENT     Sequence Label: pUC_hU6-shRKIP_BFP
FEATURES             Location/Qualifiers
     source          1..4404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        79..1257
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          310..1248
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1205..1225
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(2000..2017)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2006..2230
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2257..2361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2362..2430,2431..3222)
                     /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(2580..2599)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3393..3981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3882..3901
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4044..4292
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4044..4064
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4215..4234
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           4366..4380
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
ORIGIN
        1 tgcctactgc ctcggacttc aaggggctag aattcgtttt tttttgtatc aagtactaga
       61 actccccagt gggaattcgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      121 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
      181 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
      241 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
      301 agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc
      361 ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt
      421 cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt
      481 gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt
      541 cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct
      601 gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt
      661 atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg
      721 gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc
      781 cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg
      841 ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca
      901 gggagctcaa aatgaaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa
      961 aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg
     1021 ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg
     1081 gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca
     1141 gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc
     1201 attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgacgt
     1261 acggccacca tgagcatgtc cgaactgatt aaagagaaca tgcacatgaa actttatatg
     1321 gaaggtacgg tcgacaacca ccatttcaaa tgtacctccg aaggagaggg caaaccctac
     1381 gagggcaccc aaacgatgag aattaaagta gtagaaggtg ggccgctgcc ctttgccttt
     1441 gacattctgg caacttcttt tctctacggg agtaaaactt ttatcaacca tacgcaaggc
     1501 attccagact tctttaagca gtcatttccc gaggggttta cttgggagcg ggtcacaaca
     1561 tacgaggacg gcggcgtgct gaccgctact caggacacca gcctgcaaga cgggtgtttg
     1621 atctacaatg tgaagattcg aggcgtaaac tttacttcaa acggcccagt tatgcagaaa
     1681 aaaaccctcg gctgggaggc ttttacagag actctttacc cagccgacgg tggtttggaa
     1741 ggccgaaacg acatggcatt gaaattggtt ggcgggagcc accttatagc gaatataaag
     1801 acgacctatc ggtctaaaaa gcccgccaaa aatttgaaga tgcctggtgt atattacgtc
     1861 gactataggc ttgagagaat aaaagaagct aacaacgaaa cctatgtgga gcaacatgag
     1921 gttgcagttg cacgctactg cgatctgccg agcaaattgg ggcacaagct gaattgatca
     1981 gctagagctc gctaatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2041 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2101 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2161 gggcaggaca gcaaggggga ggattgggaa gataatagca ggcatgctgg ggatgcggtg
     2221 ggctctatgg tctagattta gggtagggga aatgtgcgcg gaacccctat ttgtttattt
     2281 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2341 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2401 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2461 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2521 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2581 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     2641 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2701 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2761 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     2821 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     2881 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     2941 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3001 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3061 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
     3121 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3181 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3241 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3301 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3361 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3421 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3481 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3541 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3601 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3661 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3721 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3781 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3841 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3901 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3961 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4021 tgctggcctt ttgctcagct agcgagggcc tatttcccat gattccttca tatttgcata
     4081 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     4141 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     4201 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     4261 ctttatatat cttgtggaaa ggacgcgact tcttaaccca acagaaggct cgagaaggta
     4321 tattgctgtt gacagtgagc gctcgtggga tggtcttgat tcatagtgaa gccacagatg
     4381 tatgaatcaa gaccatccca cgaa
//
