LOCUS       sequence-209424-        8426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRT integration of wild type TPI reporter; enables detection of 
            5'-3' decay intermediates (xrFrag) and allows detection via northern
            blot (4H binding sites).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8426)
  AUTHORS   Boehm V, Gerbracht JV, Marx MC, Gehring NH
  TITLE     Interrogating the degradation pathways of unstable mRNAs with 
            XRN1-resistant sequences.
  JOURNAL   Nat Commun. 2016 Dec 5;7:13691. doi: 10.1038/ncomms13691.
  PUBMED    27917860
REFERENCE   2  (bases 1 to 8426)
  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: "Nat 
            Commun."; date: "2016-12-5"; volume: "7:13691. doi"; pages: " 
            10.1038/ncomms13691"
COMMENT     Sequence Label: pcDNA5/FRT/TO-TPI_WT-xrRNA-4H
FEATURES             Location/Qualifiers
     source          1..8426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    589..607
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    610..628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     638..662
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(4147..4164)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4153..4377
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4510..4529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4661..4708
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4716..5738
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4716..5738
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5868..5989
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5905..5924)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5959..5978
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6038..6054)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6038..6054)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6051..6073)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6062..6078
                     /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)."
     promoter        complement(join(6086..6092,6093..6110,6111..6116))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6131..6152
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6269..6286)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6440..7028)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6520..6539)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7199..7990,7991..8059))
                     /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     7822..7841
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8060..8164)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8239..8258)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
      601 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
      661 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      721 gcctccggac tctagcgttt aaacttaagc ttgctagcgc ctcggctcca gcaccatggc
      781 gccctccagg aagttcttcg ttgggggaaa ctggaagatg aacgggcgga agcagagtct
      841 gggggagctc atcggcactc tgaacgcggc caaggtgccg gccgacaccg gtaagccctc
      901 gccgaggagg ggtctggccg ggccggggcc ggggccgggg caggagtggc agcgccctct
      961 cccgaggccc cgaggccccg aggccggtat ccgcgcggac ctgatgcagg gctgtgggac
     1021 gagggccgct ggggtccggg caggggcctc gcagccgcag ccccgtcggt gcgtcgaggg
     1081 ggcagggcgg agcacatgat gccccttgga ctatggggca ggtaaggacg ttttcttccc
     1141 ttcggcaacc tgaacgactc ccgccttcca cggaagggac cgagcccgtg ccaaacaggc
     1201 tgagcgattt gggagtgagg agccatccta ccgctttccc caacctggaa acagcaaagc
     1261 gcaaggcctc tgagtcagtt aggtctctgc cacccacggg caaaggatgc tctcctccat
     1321 cctccttcct ccctccaccg aaatcggaga gccgcgggcc tgatccaaag aggcatcccc
     1381 ttctcgttca ttccccagag gcctcaatac aaaccccagg agttggcccc tctccttttg
     1441 ctacaaatcc ttgccttgca aaggggaggt gaggatgggc tattttagaa gggaagcagg
     1501 gttgctccct ggagaatgct gagtctgtga ggtgcctatg ccgagaatag gtcgaggaaa
     1561 ttggagcccc agctgttaaa agagcagagg gcagggtgag ggccgtggcc tctcaggggt
     1621 atctggaagg ctcttcgagt tgagtgcaga cccagcctgg gctggaaaat ggacaaaggt
     1681 catcttgctg gggtgaaaag ggggagagca gaaccaagaa gaagagggtg agggctgggg
     1741 ggctccaggg cactggttag gaattgtggg gaatgaaggc tttctttagt ctcatccccc
     1801 tgtggtacca tcttgtcctc agaggtggtt tgtgctcccc ctactgccta tatcgacttc
     1861 gcccggcaga agctagatcc caagattgct gtggctgcgc agaactgcta caaagtgact
     1921 aatggggctt ttactgggga gatcaggtga gatcgaggtg gagaggggtg tgtgggaccc
     1981 ttccctcact ttcctcgttg aggggaaagc cacagggtgg gctccctgct gaaccttggc
     2041 ttcatctctt cctttagccc tggcatgatc aaagactgcg gagccacgtg ggtggtcctg
     2101 gggcactcag agagaaggca tgtctttggg gagtcagatg aggttagtag ccaagagaga
     2161 agataaggga tgtctttttc caagaaggat gtctcaccaa gtctgtttct caacagctga
     2221 ttgggcagaa agtggcccat gctctggcag agggactcgg agtaatcgcc tgcattgggg
     2281 agaagctaga tgaaagggaa gctggcatca ctgagaaggt tgttttcgag cagacaaagg
     2341 tcatcgcagg tatctctgga gaaagggacc tttgagccta tccagggcca cagagactca
     2401 gagggtaggg tcaggccctg gagcctgtct tggtccccat gctgatccag aaaaggaaaa
     2461 aggggagggg gagtgacaat ctttgcttgg ggcctatgac ttctccagcc ccaaggtaga
     2521 tgccacctgg aaatccccca atgtccacta gggggcagta ggccaccgtt cttcgtactc
     2581 cggagaacct ggctggagag ctctttcttg ttcacccttc cctccatctg tatctctgcc
     2641 ctgcagataa cgtgaaggac tggagcaagg tcgtcctggc ctatgagcct gtgtgggcca
     2701 ttggtactgg caagactgca acaccccaac aggtaaccgg gcccaggagc cctgccctca
     2761 tcccagcctg cctcaatagg tttggacaga cacagcccac atggggcaac cccttatttc
     2821 aaagacacag agaccttgaa cccagagaca gtgacttgtc caagggcatc cagtccaggg
     2881 cctggcttgg atcagagccc tggtactctg actcagtcag aaaccacact aagtgtccac
     2941 tggtgccagt gatttttcct cttagagagg cagaaaaggt cttacttagg ccagcttctt
     3001 gttctaggcc caggaagtac acgagaagct ccgaggatgg ctgaagtcca acgtctctga
     3061 tgcggtggct cagagcaccc gtatcattta tggaggtgag tggctttggt tcccggctga
     3121 ggtggagtgg gctgaggact agactgagcc ctcggacatg gaggtgggga tggggcagac
     3181 tcatcccatt cttgaccaag cccttgttct gctcccttcc caggctctgt gactggggca
     3241 acctgcaagg agctggccag ccagcctgat gtggatggct tccttgtggg tggtgcttcc
     3301 ctcaagcccg aattcgtgga catcatcaat gccaaacaat gagccccatc catcttccct
     3361 acccttcctg ccaagccagg gactcgaggt gagcaagggc gaggagctgt tcaccggggt
     3421 ggtgcccatc ctggtggact tgatagtcag gccagccggt taggctgcca ccgaaggttg
     3481 gtagacggtg ctgcctgcga ccaaccccag gaggactggg ttaccaaagc tgattctcca
     3541 cggttggaaa gcctcccaga accgtctcgg aagaggagtc cctgccaaca atggagatga
     3601 agcccgtgtc agatcgcgaa agcgccactt cgccgaggag tgcaatctgt gaggccccag
     3661 gaggactggg taaacagtcg agcaccccac cagtgcaggc tgcctatcag aaagtggtgg
     3721 ctggtgtggc taatgccctg gcccacaagt atcactaagc tcgctttctt gctgtccaat
     3781 ttgtcgagca ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat
     3841 gccctggccc acaagtatca ctaagctcgc tttcttgctg tccaatttgt cgagcacccc
     3901 accagtgcag gctgcctatc agaaagtggt ggctggtgtg gctaatgccc tggcccacaa
     3961 gtatcactaa gctcgctttc ttgctgtcca atttgtcgag caccccacca gtgcaggctg
     4021 cctatcagaa agtggtggct ggtgtggcta atgccctggc ccacaagtat cactaagctc
     4081 gctttcttgc tgtccaattt gtcgacccgg gcggccgcag agggcccgtt taaacccgct
     4141 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     4201 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     4261 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     4321 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     4381 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     4441 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4501 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4561 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4621 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     4681 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     4741 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     4801 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     4861 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     4921 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     4981 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     5041 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     5101 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     5161 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     5221 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     5281 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     5341 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     5401 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     5461 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     5521 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     5581 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     5641 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     5701 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     5761 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     5821 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     5881 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5941 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     6001 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     6061 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     6121 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     6181 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6241 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6301 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6361 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6421 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6481 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6541 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6601 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6661 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6721 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6781 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6841 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6901 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6961 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     7021 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     7081 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     7141 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7201 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7261 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7321 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7381 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7441 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7501 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7561 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7621 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7681 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7741 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7801 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7861 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7921 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7981 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     8041 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     8101 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     8161 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     8221 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     8281 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     8341 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     8401 tcgcgatgta cgggccagat atacgc
//
