LOCUS       sequence-47977-e        6765 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 6765)
  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 6765)
  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-PGK1
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             67..108
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(146..163)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    152..376
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(509..528)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    660..707
                     /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             715..1737
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    1867..1988
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1904..1923)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1958..1977
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2037..2053)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2037..2053)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2050..2072)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2061..2077
                     /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(2085..2091,2092..2109,2110..2115))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2130..2151
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2268..2285)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2439..3027)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2519..2538)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3198..3989,3990..4058))
                     /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     3821..3840
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4059..4163)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4238..4257)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4429..4808
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4809..5012
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4963..4983
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    5014..5032
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5035..5053
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5063..5087
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5187..5210
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             5214..5237
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5238..5264
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    5280..5304
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(6580..6604)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6657..6698
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(join(6755..6765,1..14))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 ttgtacaaag tggttgatat ccagcacagt ggcggccgct caagtctaga gggcccgcgg
       61 ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgct cgagtctaga
      121 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
      181 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
      241 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
      301 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
      361 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
      421 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
      481 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      541 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      601 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag
      661 aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag
      721 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc
      781 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
      841 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
      901 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggagttc
      961 agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
     1021 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct
     1081 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
     1141 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
     1201 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
     1261 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
     1321 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
     1381 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
     1441 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
     1501 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
     1561 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
     1621 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
     1681 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac
     1741 gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     1801 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     1861 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1921 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     1981 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     2041 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2101 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2161 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2221 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2281 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2341 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2401 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2461 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2521 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2581 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2641 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2701 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2761 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2821 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     2881 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2941 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3001 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3061 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3121 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3181 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3241 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3301 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3361 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3421 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3481 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3541 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3601 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3661 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3721 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3781 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3841 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3901 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3961 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4021 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4081 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4141 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     4201 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     4261 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     4321 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     4381 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     4441 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     4501 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     4561 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     4621 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     4681 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     4741 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     4801 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     4861 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg aaccaaaatc
     4921 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     4981 gtgtacggtg ggaggtctat ataagcagag ctctccctat cagtgataga gatctcccta
     5041 tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
     5101 atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc cggactctag
     5161 cgtttaaact taagcttggt accatgcacc accaccacca ccaccaccac atggactaca
     5221 aggacgacga tgacaagtac ccttatgacg tgcccgatta cgctttgaac cagatatcaa
     5281 caagtttgta caaaaaagca ggctccacca tgggaaccat gtcgctttct aacaagctga
     5341 cgctggacaa gctggacgtt aaagggaagc gggtcgttat gagagtcgac ttcaatgttc
     5401 ctatgaagaa caaccagata acaaacaacc agaggattaa ggctgctgtc ccaagcatca
     5461 aattctgctt ggacaatgga gccaagtcgg tagtccttat gagccaccta ggccggcctg
     5521 atggtgtgcc catgcctgac aagtactcct tagagccagt tgctgtagaa ctcaaatctc
     5581 tgctgggcaa ggatgttctg ttcttgaagg actgtgtagg cccagaagtg gagaaagcct
     5641 gtgccaaccc agctgctggg tctgtcatcc tgctggagaa cctccgcttt catgtggagg
     5701 aagaagggaa gggaaaagat gcttctggga acaaggttaa agccgagcca gccaaaatag
     5761 aagctttccg agcttcactt tccaagctag gggatgtcta tgtcaatgat gcttttggca
     5821 ctgctcacag agcccacagc tccatggtag gagtcaatct gccacagaag gctggtgggt
     5881 ttttgatgaa gaaggagctg aactactttg caaaggcctt ggagagccca gagcgaccct
     5941 tcctggccat cctgggcgga gctaaagttg cagacaagat ccagctcatc aataatatgc
     6001 tggacaaagt caatgagatg attattggtg gtggaatggc ttttaccttc cttaaggtgc
     6061 tcaacaacat ggagattggc acttctctgt ttgatgaaga gggagccaag attgtcaaag
     6121 acctaatgtc caaagctgag aagaatggtg tgaagattac cttgcctgtt gactttgtca
     6181 ctgctgacaa gtttgatgag aatgccaaga ctggccaagc cactgtggct tctggcatac
     6241 ctgctggctg gatgggcttg gactgtggtc ctgaaagcag caagaagtat gctgaggctg
     6301 tcactcgggc taagcagatt gtgtggaatg gtcctgtggg ggtatttgaa tgggaagctt
     6361 ttgcccgggg aaccaaagct ctcatggatg aggtggtgaa agccacttct aggggctgca
     6421 tcaccatcat aggtggtgga gacactgcca cttgctgtgc caaatggaac acggaggata
     6481 aagtcagcca tgtgagcact gggggtggtg ccagtttgga gctcctggaa ggtaaagtcc
     6541 ttcctggggt ggatgctctc agcaatattt aggatctaga cccagctttc ttgtacaaag
     6601 tggttgatat ccagcacagt ggcggccgct caagtctaga gggcccgcgg ttcgaaggta
     6661 agcctatccc taaccctctc ctcggtctcg attctacgcg tacctgatcc gcggccgcat
     6721 agataactga tccagtgtgc ctcgagatat ctagacccag ctttc
//
