LOCUS       sequence-66682-e        7006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express cGFP transcript ending in a mutant MALAT1 triple helix which
            causes the transcript to be degraded.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7006)
  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 7006)
  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' Mut U1/U2 Sense
FEATURES             Location/Qualifiers
     source          1..7006
                     /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(3439..3458)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3440..3458)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3465..3482)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3465..3481)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3474..3496)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3622..4050
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3709..3728)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3919..3940
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4394..5188
                     /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(4448..4467)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5058..5077
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(5206..5274,5275..6066)
                     /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(5424..5443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6237..6825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6726..6745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6979..6996
                     /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 gccgcgattc gtcagtaggg ttgtaaaggt ttaaaaattc
     2461 ctgagaaaac aaccttttgt tttctcaggt ttaaaaattt ggcctttccc tagctttaaa
     2521 aaaaaaaaag caaaagacgc tggtggctgg cactcctggt ttccaggacg gggttcaagt
     2581 ccctgcggtg tctttgcttg cggccgcgct gtgcggtagg cattgaggca gccagcgcag
     2641 gggcttctgc tgagggggca ggcggagctt gaggaaaccg cagataagtt tttttctctt
     2701 tgaaagatag agattaatac aactacttaa aaaatatagt caataggtta ctaagatatt
     2761 gcttagcgtt aagtttttaa cgtaatttta atagcttaag attttaagag aaaatatgaa
     2821 gacttagaag agtagcatga ggaaggaaaa gataaaaggt ttctaaaaca tgacggaggt
     2881 tgagatgaag cttcttcatg gagtaaaaaa tgtatttaaa agaaaattga gagaaaggac
     2941 tacagagccc cgaattaata ccaatagaag ggcaatgctt ttagattaaa atgaaggtga
     3001 cttaaacagc ttaaagttta gtttaaaagt tgtaggtgat taaaataatt tgaaggcgat
     3061 cttttaaaaa gagattaaac cgaaggtgat taaaagacct tgaaatccat gacgcaggga
     3121 gaattgcgtc atttaaagcc tagttaacgc atttactaaa cgcagacgaa aatggaaaga
     3181 ttaattggga gtggtaggat gaaacaattt ggagaagata gaagtttgaa gtggaaaact
     3241 ggaagacaga agtacgggaa ggcgaagaaa agaatagaga agatagggaa attagaagat
     3301 aaaaacatac ttttagaaga aaaaagataa atttaaacct gaaaagtagg aagcagaaga
     3361 aaaaagacaa gctaggaaac aaaaagctaa gggcaaaatg tacaaactct cgagcatgca
     3421 tctagagggc ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta
     3481 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     3541 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     3601 cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3661 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3721 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3781 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3841 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3901 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3961 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4021 agctgattta acaaaaattt aacgcgaatt ttaacaaaat tcagggcgca agggctgcta
     4081 aaggaagcgg aacacgtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt
     4141 cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc aggtagcttg
     4201 cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga
     4261 attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc
     4321 tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag agacaggatg
     4381 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4441 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4501 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4561 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4621 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4681 agtgccgggg caggatctcc tgtcatccca ccttgctcct gccgagaaag tatccatcat
     4741 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4801 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4861 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4921 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4981 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     5041 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     5101 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     5161 ctatcgcctt cttgacgagt tcttctgaat tgaaaaagga agagtatgag tattcaacat
     5221 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5281 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5341 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5401 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5461 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5521 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5581 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5641 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5701 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5761 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5821 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5881 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5941 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6001 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6061 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6121 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6181 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6241 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6301 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6361 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6421 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6481 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6541 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6601 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6661 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6721 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6781 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6841 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6901 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6961 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
