LOCUS       sequence-191088-        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  JOURNAL   Unpublished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            "Unpublished"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        38..286
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     209..228
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        370..673
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        674..877
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     828..848
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      933..942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             939..1646
                     /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(1084..1102)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             1656..1685
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1810..1931
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1847..1866)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1901..1920
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1938..2393)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2075..2096)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2287..2306
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2420..2524
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2526..2883
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2526..2717
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2549..2569)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2734..2869
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2918..3712
                     /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             2918..3712
                     /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(2972..2991)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3582..3601
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3900..3919)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3944..3991
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4320..4908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4809..4828
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttattaataa gatctccggt ccaaggtcgg gcaggaagag ggcctatttc ccattattcc
       61 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
      121 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
      181 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      241 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccaagt cttcaagaag
      301 acattttttt actagttaat agtaatcaat tacggggtca ttagttcata gcccatatat
      361 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      421 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      481 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      541 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      601 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      661 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      721 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      781 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      841 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg
      901 ctagcgctac cggactcaga tccataccgg tcgccaccat ggtgagcaag ggcgaggagg
      961 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     1021 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     1081 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     1141 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     1201 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     1261 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     1321 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaaaaag accatgggct
     1381 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     1441 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     1501 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     1561 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     1621 ccggcggcat ggacgagctg tacaaggccg cagccgaaca aaagttgatt tctgaagaag
     1681 atttgtaagc ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta
     1741 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     1801 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     1861 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     1921 aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     1981 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     2041 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     2101 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     2161 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     2221 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     2281 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     2341 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     2401 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2461 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2521 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     2581 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     2641 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2701 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     2761 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     2821 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     2881 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     2941 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3001 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3061 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     3121 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3181 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3241 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3301 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3361 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3421 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     3481 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3541 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     3601 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     3661 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     3721 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     3781 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3841 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     3901 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     3961 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     4021 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     4081 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     4141 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     4201 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     4261 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4321 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4381 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4441 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     4501 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4561 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4621 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4681 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4741 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4801 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4861 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4921 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4981 ttatcccctg attctgtgga taaccgtatt accgccatgc attag
//
