LOCUS       sequence-66684-e        6955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express cGFP transcript ending in a mutant MALAT1 triple helix that 
            causes the transcript to be stable but poorly translated.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6955)
  AUTHORS   Wilusz JE, JnBaptiste CK, Lu LY, Kuhn CD, Joshua-Tor L, Sharp PA
  TITLE     A triple helix stabilizes the 3' ends of long noncoding RNAs that 
            lack poly(A) tails.
  JOURNAL   Genes Dev. 2012 Nov 1;26(21):2392-407. doi: 10.1101/gad.204438.112. 
            Epub 2012 Oct 16.
  PUBMED    23073843
REFERENCE   2  (bases 1 to 6955)
  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: 
            "10.1101/gad.204438.112"; journalName: "Genes Dev"; date: 
            "2012-11-1- 1"; volume: "26"; issue: "21"; pages: "2392-407"
COMMENT     Sequence Label: pCRII-TOPO CMV-cGFP-mMALAT1_3' Comp.15 Sense
FEATURES             Location/Qualifiers
     source          1..6955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1075..1453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1454..1657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1608..1628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(3388..3407)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3389..3407)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3414..3431)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3414..3430)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3423..3445)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3571..3999
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3658..3677)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3868..3889
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4343..5137
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4397..4416)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5007..5026
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(5155..5223,5224..6015)
                     /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(5373..5392)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6186..6774
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6675..6694
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6928..6945
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tctttaaaag gccacttgaa
      301 ctcgctttcc atggcgattt gccttgtgag cactttcagg agagcctgga agctgaaaaa
      361 cggtagaaaa atttccgtgc gggccgtggg gggctggcgg caactggggg gccgcagatc
      421 agagtgggcc actggcagcc aacggccccc ggggctcagg cggggagcag ctctgtggtg
      481 tgggattgag gcgttttcca agagtgggtt ttcacgtttc taagatttcc caagcagaca
      541 gcccgtgctg ctccgatttc tcgaacaaaa aagcaaaacg tgtggctgtc ttgggagcaa
      601 gtcgcaggac tgcaagcagt tgggggagaa agtccgccat tttgccactt ctcaaccgtc
      661 cctgcaaggc tggggctcag ttgcgtaatg gaaagtaaag ccctgaacta tcacacttta
      721 atcttccttc aaaaggtggt aaactatacc tactgtccct caagagaaca caagaagtgc
      781 tttaagaggt attttaaaag ttccgggggt tttgtgaggt gtttgatgac ccgtttaaaa
      841 tatgatttcc atgtttcttt tgtctaaagt ttgcagctca aatctttcca cacgctagta
      901 atttaagtat ttctgcatgt gtagtttgca ttcaagttcc ataagctgtt aagaaaaatc
      961 tagaaaagta aaactagaac ctatttttaa ccgaagaact actttttgcc tccctcacaa
     1021 aggcggcgga aggtgatcga attccggtga tgcgagttgt tctccgtcgg atccacattg
     1081 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     1141 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     1201 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     1261 ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac atcaagtgta
     1321 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     1381 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     1441 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     1501 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     1561 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     1621 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
     1681 tgcttactgg cgatatcatg gccagcaagg gcgaggagct gttcaccggc gtggtgccca
     1741 tcctggtgga gctggacggc gatgtgaatg gccacaagtt cagcgtgagc ggcgagggcg
     1801 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1861 ctgtgccctg gcccaccctg gtgaccaccc tgagctacgg cgtgcagtgc ttctcacgct
     1921 accccgatca catgaagcag cacgacttct tcaagagcgc catgcctgag ggctacatcc
     1981 aggagcgcac catcttcttc gaggatgacg gcaactacaa gtcgcgcgcc gaggtgaagt
     2041 tcgagggcga taccctggtg aatcgcatcg agctgaccgg caccgatttc aaggaggatg
     2101 gcaacatcct gggcaataag atggagtaca actacaacgc ccacaatgtg tacatcatga
     2161 ccgacaaggc caagaatggc atcaaggtga acttcaagat ccgccacaac atcgaggatg
     2221 gcagcgtgca gctggccgac cactaccagc agaatacccc catcggcgat ggccctgtgc
     2281 tgctgcccga taaccactac ctgtccaccc agagcgccct gtccaaggac cccaacgaga
     2341 agcgcgatca catgatctac ttcggcttcg tgaccgccgc cggcatcacc cacggcatgg
     2401 acgagctgta caagtgagcg gccgccccaa tttttctttt gaattctcta gagaattctt
     2461 ttgctttttc ttcaaaaagc aaaagacgct ggtggctggc actcctggtt tccaggacgg
     2521 ggttcaagtc cctgcggtgt ctttgcttgc ggccgcgctg tgcggtaggc attgaggcag
     2581 ccagcgcagg ggcttctgct gagggggcag gcggagcttg aggaaaccgc agataagttt
     2641 ttttctcttt gaaagataga gattaataca actacttaaa aaatatagtc aataggttac
     2701 taagatattg cttagcgtta agtttttaac gtaattttaa tagcttaaga ttttaagaga
     2761 aaatatgaag acttagaaga gtagcatgag gaaggaaaag ataaaaggtt tctaaaacat
     2821 gacggaggtt gagatgaagc ttcttcatgg agtaaaaaat gtatttaaaa gaaaattgag
     2881 agaaaggact acagagcccc gaattaatac caatagaagg gcaatgcttt tagattaaaa
     2941 tgaaggtgac ttaaacagct taaagtttag tttaaaagtt gtaggtgatt aaaataattt
     3001 gaaggcgatc ttttaaaaag agattaaacc gaaggtgatt aaaagacctt gaaatccatg
     3061 acgcagggag aattgcgtca tttaaagcct agttaacgca tttactaaac gcagacgaaa
     3121 atggaaagat taattgggag tggtaggatg aaacaatttg gagaagatag aagtttgaag
     3181 tggaaaactg gaagacagaa gtacgggaag gcgaagaaaa gaatagagaa gatagggaaa
     3241 ttagaagata aaaacatact tttagaagaa aaaagataaa tttaaacctg aaaagtagga
     3301 agcagaagaa aaaagacaag ctaggaaaca aaaagctaag ggcaaaatgt acaaactctc
     3361 gagcatgcat ctagagggcc caattcgccc tatagtgagt cgtattacaa ttcactggcc
     3421 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     3481 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     3541 caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg
     3601 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3661 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3721 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3781 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3841 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3901 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     3961 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaatt cagggcgcaa
     4021 gggctgctaa aggaagcgga acacgtagaa agccagtccg cagaaacggt gctgaccccg
     4081 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca
     4141 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag
     4201 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa
     4261 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga
     4321 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4381 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4441 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4501 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4561 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4621 attgggcgaa gtgccggggc aggatctcct gtcatcccac cttgctcctg ccgagaaagt
     4681 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4741 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4801 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4861 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4921 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4981 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5041 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5101 catcgccttc tatcgccttc ttgacgagtt cttctgaatt gaaaaaggaa gagtatgagt
     5161 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5221 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5281 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5341 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5401 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5461 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5521 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5581 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5641 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5701 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5761 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5821 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5881 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5941 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6001 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6061 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6121 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6181 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6241 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6301 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6361 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6421 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6481 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6541 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6601 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6661 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6721 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6781 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6841 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     6901 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
