LOCUS       sequence-146530-        5025 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.
            CMV driven mCherry visible marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5025)
  TITLE     John Rossi lab small RNA expression plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5025)
  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            "Unpublished"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: pJR255
FEATURES             Location/Qualifiers
     source          1..5025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        41..289
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     212..231
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        373..676
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        677..880
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     831..851
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      936..945
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             942..1649
                     /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(1087..1105)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             1659..1688
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1813..1934
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1850..1869)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1904..1923
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1941..2396)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2078..2099)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2290..2309
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2423..2527
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2529..2886
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2529..2720
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2552..2572)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2737..2872
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2921..3715
                     /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             2921..3715
                     /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(2975..2994)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3585..3604
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3903..3922)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3947..3994
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4323..4911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4812..4831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa taagatctcc ggtccaaggt cgggcaggaa gagggcctat ttcccattat
       61 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
      121 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
      181 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
      241 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacacca agtcttcaag
      301 aagacatttt tttactagtt aatagtaatc aattacgggg tcattagttc atagcccata
      361 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      421 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      481 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      541 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      601 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      661 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      721 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      781 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      841 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
      901 ccgctagcgc taccggactc agatccatac cggtcgccac catggtgagc aagggcgagg
      961 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     1021 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     1081 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     1141 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     1201 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     1261 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     1321 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaaa aagaccatgg
     1381 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     1441 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     1501 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     1561 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     1621 ccaccggcgg catggacgag ctgtacaagg ccgcagccga acaaaagttg atttctgaag
     1681 aagatttgta agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     1741 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     1801 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     1861 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     1921 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     1981 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     2041 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     2101 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     2161 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     2221 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     2281 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     2341 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     2401 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2461 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2521 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     2581 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2641 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2701 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2761 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2821 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     2881 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     2941 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3001 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3061 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     3121 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3181 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3241 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3301 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3361 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3421 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     3481 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3541 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3601 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3661 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3721 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3781 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3841 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     3901 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     3961 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     4021 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     4081 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     4141 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     4201 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4261 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4321 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4381 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4441 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4501 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4561 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4621 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4681 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4741 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4801 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4861 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4921 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4981 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
