LOCUS       sequence-239355-        7187 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 7187)
  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 7187)
  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-MYEF2
FEATURES             Location/Qualifiers
     source          1..7187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..49)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    38..262
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(395..414)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    546..593
                     /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             601..1623
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             601..1623
                     /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    1753..1874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1790..1809)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1844..1863
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1923..1939)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1923..1939)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1936..1958)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1947..1963
                     /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(1971..1977,1978..1995,1996..2001))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2016..2037
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2154..2171)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2325..2913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2405..2424)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3084..3875,3876..3944))
                     /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     3707..3726
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3945..4049)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4124..4143)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4315..4694
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4695..4898
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4849..4869
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4900..4918
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4921..4939
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4949..4973
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5073..5096
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             5100..5123
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5124..5150
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    5166..5190
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(7021..7045)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             7098..7139
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
ORIGIN
        1 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
       61 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
      121 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
      181 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
      241 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
      301 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      361 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      421 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      481 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      541 acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc
      601 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
      661 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
      721 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
      781 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
      841 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
      901 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
      961 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
     1021 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
     1081 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
     1141 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
     1201 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg
     1261 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
     1321 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg
     1381 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
     1441 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
     1501 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt
     1561 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
     1621 tagcacgtac tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     1681 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     1741 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     1801 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     1861 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     1921 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1981 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     2041 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2101 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2161 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2221 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2281 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2341 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2401 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2461 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2521 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2581 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2641 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2701 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2761 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2821 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2881 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2941 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3001 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3061 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3121 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3181 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3241 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3301 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3361 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3421 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3481 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3541 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3601 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3661 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3721 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3781 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3841 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3901 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3961 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4021 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4081 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     4141 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     4201 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     4261 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
     4321 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     4381 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     4441 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     4501 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     4561 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     4621 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     4681 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     4741 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     4801 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     4861 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
     4921 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
     4981 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
     5041 ctctagcgtt taaacttaag cttggtacca tgcaccacca ccaccaccac caccacatgg
     5101 actacaagga cgacgatgac aagtaccctt atgacgtgcc cgattacgct ttgaaccaga
     5161 tatcaacaag tttgtacaaa aaagcagaag ggccgtcaag gcccaccatg gcggacgcca
     5221 acaaggccga ggtgcccggg gccactggtg gcgacagccc gcacctgcag cccgcagagc
     5281 cgccgggcga gccgcggcga gagccgcacc ccgcggaggc ggagaagcag cagccgcagc
     5341 acagcagcag ctccaatggc gttaaaatgg agaatgatga atcagcaaaa gaagagaaat
     5401 ctgacttaaa ggaaaaatct acaggaagta agaaggccaa tagatttcat ccttattcaa
     5461 aagacaagaa ttcgggcact ggagaaaaga agggtccaaa tcgtaacaga gttttcatta
     5521 gcaacatccc atatgacatg aaatggcaag ctattaaaga tctaatgaga gagaaagttg
     5581 gtgaggttac atacgtggag ctctttaagg atgcggaagg aaaatcaagg ggttgtggtg
     5641 tggttgaatt caaagatgaa gaatttgtaa agaaagccct agaaactatg aacaaatatg
     5701 atcttagtgg aagacccctt aatattaaag aggatcctga tggagaaaat gctcgtaggg
     5761 cattgcagcg aacaggagga tcatttccag gaggacacgt ccctgatatg ggatcagggt
     5821 tgatgaattt accaccttcc atactcaata atccaaacat tcctcctgaa gtcatcagta
     5881 atttgcaggc cggtagactt ggttccacaa tttttgttgc caatcttgac ttcaaagttg
     5941 gttggaagaa gctaaaggaa gtgttcagca tagctggaac tgtgaagcgg gcagatatta
     6001 aagaagacaa agatggcaag agcagaggaa tgggcactgt cacttttgag caagcaattg
     6061 aagcagttca agcaatttct atgttcaatg ggcagttttt atttgataga cctatgcatg
     6121 tgaaaatgga tgacaagtct gttcctcatg aagagtaccg ttcacatgat ggtaaaacac
     6181 cacaattacc acgtggtctt ggaggcattg ggatgggact tggtccgggt ggacagccta
     6241 ttagtgccag ccagttgaac ataggtggag taatgggaaa tttaggtcca ggtggtatgg
     6301 gaatggatgg tccaggtttt ggaggaatga atagaattgg aggaggaata gggtttggtg
     6361 gtctggaagc aatgaatagc atgggaggat ttggaggagt tggccgaatg ggagagctgt
     6421 accgtggtgc gatgactagt agcatggagc gagattttgg acgtggtgat attggaataa
     6481 atcgaggctt tggagattcc tttggtagac ttggcagtgc aatgattgga gggtttgcag
     6541 gaagaatagg atcttctaac atgggtccag taggatctgg aataagtggt ggaatgggta
     6601 gcatgaacag tgtgactgga ggaatgggga tgggactgga ccggatgagt tccagctttg
     6661 atagaatggg accaggtata ggagctatac tggaaaggag catcgatatg gatcgaggat
     6721 ttttatcggg tccaatggga agcggaatga gagagagaat aggctccaaa ggcaaccaga
     6781 tatttgtcag aaatctacct tttgacttga cttggcagaa actaaaagag aaattcagtc
     6841 agtgtggtca tgtaatgttt gcagaaataa aaatggagaa tggaaagtca aaaggctgtg
     6901 gaacagtcag atttgactcc ccagaatcag ctgaaaaagc ctgcagaata atgaatggca
     6961 taaaaatcag tggcagagaa attgatgttc gcttggatcg taatgcatag ggcctcatgg
     7021 gcccagcttt cttgtacaaa gtggttgata tccagcacag tggcggccgc tcaagtctag
     7081 agggcccgcg gttcgaaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc
     7141 gtacctgatc cgcggccgca tagataactg atccagtgtg cctcgag
//
