LOCUS       sequence-489382-        6742 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6742)
  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 6742)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 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-ALKBH5
FEATURES             Location/Qualifiers
     source          1..6742
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      286..874
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     775..794
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1028..1045
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1162..1183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1198..1203,1204..1221,1222..1228)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1236..1252
                     /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     1241..1263
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1260..1276
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1260..1276
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1325..1446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1336..1355)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1390..1409
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1576..2598)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(1576..2598)
                     /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
                     "
     protein_bind    complement(2606..2653)
                     /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)."
     primer_bind     2785..2804
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2937..3161
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3150..3167
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3247..3288)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    3341..3365
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4751..4775)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(4791..4817)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4818..4841)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(4845..4868)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4968..4992)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5002..5020
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5023..5041
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(5043..5246)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5072..5092)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5247..5626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5798..5817
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5892..5996
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5997..6065,6066..6742,1..115)
                     /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(6215..6234)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
       61 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
      121 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
      181 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
      241 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
      301 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
      361 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
      421 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
      481 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      541 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      601 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      661 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      721 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      781 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      841 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      901 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
      961 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     1021 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     1081 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     1141 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     1201 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     1261 aggaaacagc tatgaccatg attacgccaa gctctagcta gaggtcgacg gtatacagac
     1321 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     1381 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     1441 caagttgggg tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc
     1501 ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa
     1561 atctcgtagt acgtgctatt cctttgccct cggacgagtg ctggggcgtc ggtttccact
     1621 atcggcgagt acttctacac agccatcggt ccagacggcc gcgcttctgc gggcgatttg
     1681 tgtacgcccg acagtcccgg ctccggatcg gacgattgcg tcgcatcgac cctgcgccca
     1741 agctgcatca tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa gaccaatgcg
     1801 gagcatatac gcccggagcc gcggcgatcc tgcaagctcc ggatgcctcc gctcgaagta
     1861 gcgcgtctgc tgctccatac aagccaacca cggcctccag aagaagatgt tggcgacctc
     1921 gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg accgctgtta tgcggccatt
     1981 gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg aggtgccgga cttcggggca
     2041 gtcctcggcc caaagcatca gctcatcgag agcctgcgcg acggacgcac tgacggtgtc
     2101 gtccatcaca gtttgccagt gatacacatg gggatcagca atcgcgcata tgaaatcacg
     2161 ccatgtagtg tattgaccga ttccttgcgg tccgaatggg ccgaacccgc tcgtctggct
     2221 aagatcggcc gcagcgatcg catccatggc ctccgcgacc ggctgcagaa cagcgggcag
     2281 ttcggtttca ggcaggtctt gcaacgtgac accctgtgca cggcgggaga tgcaataggt
     2341 caggctctcg ctgaattccc caatgtcaag cacttccgga atcgggagcg cggccgatgc
     2401 aaagtgccga taaacataac gatctttgta gaaaccatcg gcgcagctat ttacccgcag
     2461 gacatatcca cgccctccta catcgaagct gaaagcacga gattcttcgc cctccgagag
     2521 ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc
     2581 ggtgagttca ggctttttgg ccaaggaagt tcctatactt tctagagaat aggaacttcg
     2641 gaataggaac ttctaggtac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     2701 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     2761 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     2821 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     2881 acagggcgcg tggggatacc ccctagagcc ccagctggtt ctttccgcct cagaagccat
     2941 agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc ccttgctgtc
     3001 ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg atgcaatttc
     3061 ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg aaggcacggg
     3121 ggaggggcaa acaacagatg gctggcaact agaaggcaca gtcgaggctg atcagcgggt
     3181 ttaaacgggc cctctagact cgaggcacac tggatcagtt atctatgcgg ccgcggatca
     3241 ggtacgcgta gaatcgagac cgaggagagg gttagggata ggcttacctt cgaaccgcgg
     3301 gccctctaga cttgagcggc cgccactgtg ctggatatca accactttgt acaagaaagc
     3361 tgggtctaga tctattgatg ccaacagcct ttccatccag gaggaatacc aagaatatga
     3421 gaacctaggt cctggccaat tctattcttc agccctaaca ggcaacaaaa ccaaggaaaa
     3481 atatatagaa tcaaaaaaaa caaaaaacaa aaaacaaaaa ccccccaatg aacaaaaaca
     3541 aaaccctcaa aacaaaagga ggggcaacta cgtaaggatg agccagagtt cccagggctg
     3601 cctgctgcct cagagcagtc ctctgaggac tcgtatgact tgcgccagta gttctcagag
     3661 ctgaagctac ccctccgccg gtgtgtgggc agcagcaccg agcgccggtt ctcttccttg
     3721 tccatctcca gaatccgtgg cctgtgggca gcatcagggt ctgccttgcg gtgggaccgc
     3781 ttgggtttca gagcagggtc cctgttgttt cctgacaggc gatctgaagc atagctgggt
     3841 ggtaacacgg agctgctcag ggactttgtt tccaaccggg gtgcatctaa tcttgtcttc
     3901 ctgaggatga tgactgctcg gcgctccttg atgtcctgag gccgtatgca gtgagtgatt
     3961 tcatcagcag catatccact gagcacagtc acgcttcccc tgcgcaccgg cagggaaagc
     4021 actggttccg acacccgaat aggcttgaac tggaacttgc agccgaagca cagcgcagag
     4081 tcgctaaaga aggacacgga cacgatgggg cgctcgaaga tgtggatggg gtccacgtga
     4141 gacacgatgc agccgccggg ctggtagtcg ttgatgacgg cgctgttgac gaagccctcg
     4201 gggatgacgc ggtgctccac cagcttttgg atcaccagct ggtgcaccca ctcggggatc
     4261 tcgtccacgt cgcccggcgg gtagaggcgc tcctggccgg gcccgcgctt ctgcagctgg
     4321 gcgccgtaag tgtagccttc gccgaagaag tacttgttgc gcagtggggc ccggtccacc
     4381 gtgtgctcgt tgtacaggcc cttctcagcg cgggacacca cctcgtcaat gcgggcctcg
     4441 atcttggcgc actcgtcctg gctgaagagg cgcatctggc ggatgccgct cttcaccttg
     4501 cgcgcctcct cctccttctg cagctgctgc tcctcatagt cgctgcgctc ggggtccgag
     4561 tcctcctgat acttgcgctt ggccccggac acagggtaag gttcggcggc agcggcggct
     4621 gcggcagcgg cggcggcctc ccggctgccc gccttatagt tgtcccggga cgtcatggac
     4681 ttgagcttct cacgcaggtc cgtgtagccg ctggcggccg ccatgtcgac tgaattggtt
     4741 cccatggtgg agcctgcttt tttgtacaaa cttgttgata tctggttcaa agcgtaatcg
     4801 ggcacgtcat aagggtactt gtcatcgtcg tccttgtagt ccatgtggtg gtggtggtgg
     4861 tggtggtgca tggtaccaag cttaagttta aacgctagag tccggaggct ggatcggtcc
     4921 cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat ctgacggttc
     4981 actaaacgag ctcgtcgacg atctctatca ctgataggga gatctctatc actgataggg
     5041 agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg
     5101 gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca
     5161 ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca
     5221 ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac
     5281 tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta
     5341 ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag
     5401 tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt
     5461 actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca
     5521 ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat
     5581 gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg cgtatatctg
     5641 gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaattg
     5701 tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc
     5761 aacacacaag cagggagcag atactggctt aactatgcgg catcagagca gattgtactg
     5821 agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt ggcacttttc
     5881 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5941 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6001 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6061 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6121 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6181 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     6241 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6301 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6361 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6421 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6481 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6541 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6601 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6661 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6721 gtatcattgc agcactgggg cc
//
