LOCUS       sequence-191096-        4974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expressing small noncoding RNAs from U6 promoter. One step 
            cloning of oiigonucleotide pairs containing CACC and AAAA overhangs.
            Ef1a driven mCherry visible marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4974)
  TITLE     John Rossi lab small RNA expression plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4974)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pJR288
FEATURES             Location/Qualifiers
     source          1..4974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..283
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     206..225
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        332..543
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             556..824
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      879..888
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             885..1592
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1030..1048)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             1602..1631
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1756..1877
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1793..1812)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1847..1866
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1884..2339)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2021..2042)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2233..2252
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2366..2470
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2472..2829
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2472..2663
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2495..2515)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2680..2815
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2864..3658
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2864..3658
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2918..2937)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3528..3547
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3846..3865)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3890..3937
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4266..4854
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4755..4774
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaataagat ctccggtcca aggtcgggca ggaagagggc ctatttccca ttattccttc
       61 atatttgcat atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa
      121 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
      181 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
      241 cgatttcttg gctttatata tcttgtggaa aggacgaaac accaagtctt caagaagaca
      301 tttttttact agtgctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
      361 gagaagttgg ggggaggggt cggcaattga acgggtgcct agagaaggtg gcgcggggta
      421 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
      481 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
      541 cagctgaagc ttcgaggggc tcgcatctct ccttcacgcg cccgccgccc tacctgaggc
      601 cgccatccac gccggttgag tcgcgttctg ccgcctcccg cctgtggtgc ctcctgaact
      661 gcgtccgccg tctaggtaag tttaaagctc aggtcgagac cgggcctttg tccggcgctc
      721 ccttggagcc tacctagact cagccggctc tccacgcttt gcctgaccct gcttgctcaa
      781 ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca gatccaagct gtgaccggcg
      841 cctacgctag cgctaccgga ctcagatcca taccggtcgc caccatggtg agcaagggcg
      901 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
      961 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1021 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1081 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     1141 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     1201 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     1261 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aaaaagacca
     1321 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     1381 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1441 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     1501 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     1561 actccaccgg cggcatggac gagctgtaca aggccgcagc cgaacaaaag ttgatttctg
     1621 aagaagattt gtaagcggcc gcgactctag atcataatca gccataccac atttgtagag
     1681 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     1741 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1801 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     1861 ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     1921 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1981 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     2041 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2101 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2161 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     2221 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     2281 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     2341 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2401 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2461 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     2521 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2581 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2641 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2701 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2761 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2821 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     2881 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     2941 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3001 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     3061 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3121 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3181 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3241 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3301 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3361 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     3421 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3481 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3541 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3601 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     3661 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     3721 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     3781 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     3841 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     3901 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     3961 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     4021 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     4081 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     4141 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4201 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4261 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4321 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4381 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4441 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4501 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4561 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4621 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4681 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4741 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4801 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4861 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4921 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta gtta
//
