LOCUS       sequence-326090-        7449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7449)
  AUTHORS   Baltz AG, Munschauer M, Schwanhausser B, Vasile A, Murakawa Y, 
            Schueler M, Youngs N, Penfold-Brown D, Drew K, Milek M, Wyler E, 
            Bonneau R, Selbach M, Dieterich C, Landthaler M
  TITLE     The mRNA-Bound Proteome and Its Global Occupancy Profile on 
            Protein-Coding Transcripts.
  JOURNAL   Mol Cell. 2012 Jun 8;46(5):674-90.
  PUBMED    22681889
REFERENCE   2  (bases 1 to 7449)
  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: "Mol Cell.";
            date: "2012-06-8"; volume: "46(5)"; pages: "674-90"
COMMENT     Sequence Label: pFRT/TO/HIS/FLAG/HA-HNRNPR
FEATURES             Location/Qualifiers
     source          1..7449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             759..782
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             786..809
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             810..836
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    852..876
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2794..2818)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2871..2912
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(2969..2993)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3046..3087
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3167..3184)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3173..3397
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3530..3549)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3681..3728
                     /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             3736..4758
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3736..4758
                     /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    4888..5009
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4925..4944)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4979..4998
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5058..5074)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5058..5074)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5071..5093)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5082..5098
                     /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(5106..5112,5113..5130,5131..5136))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5151..5172
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5289..5306)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5460..6048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5540..5559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6219..7010,7011..7079))
                     /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     6842..6861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7080..7184)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7259..7278)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagcttg gtaccatgca ccaccaccac caccaccacc
      781 acatggacta caaggacgac gatgacaagt acccttatga cgtgcccgat tacgctttga
      841 accagatatc aacaagtttg tacaaaaaag caggctccac catggctaat caggtgaatg
      901 gtaatgcggt acagttaaaa gaagaggaag aaccaatgga tacttccagt gtaactcaca
      961 cagaacacta caagacactg atagaggcag gcctcccaca gaaggtggca gaaagacttg
     1021 atgaaatatt tcagacagga ttggtagctt atgtcgatct tgatgaaaga gcaattgatg
     1081 ctctcaggga atttaatgaa gaaggagctc tgtctgtact acagcagttc aaggaaagtg
     1141 acttatcaca tgttcagaac aaaagtgcat ttttatgtgg agttatgaag acctacaggc
     1201 agagagagaa acaggggagc aaggtgcaag agtccacaaa gggacctgat gaagcgaaga
     1261 tcaaggcctt gcttgagaga actggttata ctctggatgt aaccacagga cagaggaagt
     1321 atggtggtcc tccaccagac agtgtgtact ctggcgtgca acctggaatt ggaacggagg
     1381 tatttgtagg caaaatacca agggatttat atgaggatga gttggtgccc ctttttgaga
     1441 aggccggacc catttgggat ctacgtctta tgatggatcc actgtccggt cagaatagag
     1501 ggtatgcatt tatcaccttc tgtggaaagg aagctgcaca ggaagccgtg aaactgtgtg
     1561 acagctatga aattcgccct ggtaaacacc ttggagtgtg catttctgtg gcaaacaaca
     1621 gactttttgt tggatccatt ccgaagaata agactaaaga aaacattttg gaagaattca
     1681 gtaaagtcac aggtttaaca gagggtttgg tggacgttat tctctatcat caacccgatg
     1741 acaaaaagaa gaatcggggg ttctgcttcc ttgaatatga ggatcacaag tcagcagcac
     1801 aagccagacg ccggctgatg agtggaaaag taaaagtgtg gggaaatgta gttacagttg
     1861 aatgggctga ccctgtggaa gaaccagatc cagaagtcat ggctaaggta aaagttttgt
     1921 ttgtgagaaa cttggctact acggtgacag aagaaatatt ggaaaagtca ttttctgaat
     1981 ttggaaaact cgaaagagta aagaagttga aagattatgc atttgttcat tttgaagaca
     2041 gaggagcagc tgttaaggct atggatgaaa tgaatggcaa agaaatagaa ggggaagaaa
     2101 ttgaaatagt cttagccaag ccaccagaca agaaaaggaa agagcgccaa gctgctagac
     2161 aggcctccag aagcactgcg tatgaagatt attactacca ccctcctcct cgcatgccac
     2221 ctccaattag aggtcggggt cgtggtgggg ggagaggtgg atatggctac cctccagatt
     2281 actacggcta tgaagattac tatgatgatt actatggtta tgattatcac gactatcgtg
     2341 gaggctatga agatccctac tacggctatg atgatggcta tgcagtaaga ggaagaggag
     2401 gaggaagggg agggcgaggt gctccaccac caccaagggg gaggggagca ccacctccaa
     2461 gaggtagagc tggctattca cagagggggg cacctttggg accaccaaga ggctctaggg
     2521 gtggcagagg gggtcctgct caacagcaga gaggccgtgg ttcccgtgga tctcggggca
     2581 atcgtggggg caatgtagga ggcaagagaa aggcagatgg gtacaaccag cctgattcca
     2641 agcgtcgtca gaccaacaac caacagaact ggggttccca acccatcgct cagcagccgc
     2701 ttcagcaagg tggtgactat tctggtaact atggttacaa taatgacaac caggaatttt
     2761 atcaggatac ttatgggcaa cagtggaagt aggacccagc tttcttgtac aaagtggttg
     2821 atatccagca cagtggcggc cgctcaagtc tagagggccc gcggttcgaa ggtaagccta
     2881 tccctaaccc tctcctcggt ctcgattcta cgcgtacctg atccgcggcc gcatagataa
     2941 ctgatccagt gtgcctcgag atatctagac ccagctttct tgtacaaagt ggttgatatc
     3001 cagcacagtg gcggccgctc aagtctagag ggcccgcggt tcgaaggtaa gcctatccct
     3061 aaccctctcc tcggtctcga ttctacgcgt acctgatccg cggccgcata gataactgat
     3121 ccagtgtgcc tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     3181 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3241 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3301 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3361 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3421 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3481 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3541 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3601 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3661 ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata
     3721 ggaacttcct tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc
     3781 gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct
     3841 ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt
     3901 ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa
     3961 gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag
     4021 ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg
     4081 gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc
     4141 ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat
     4201 ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag
     4261 gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac
     4321 gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac
     4381 tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg
     4441 ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt
     4501 gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag
     4561 agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc
     4621 gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc
     4681 tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt
     4741 ccgagggcaa aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa
     4801 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4861 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4921 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4981 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     5041 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     5101 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5161 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5221 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5281 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5341 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5401 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5461 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5521 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5581 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5641 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5701 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5761 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5821 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5881 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5941 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6001 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6061 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6121 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6181 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6241 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6301 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6361 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6421 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6481 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6541 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6601 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6661 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6721 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6781 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6841 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6901 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6961 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7021 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7081 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7141 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7201 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     7261 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     7321 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     7381 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     7441 atacgcgtt
//
