LOCUS       sequence-456582-        6743 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Flag-tagged human AF9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6743)
  AUTHORS   Takahashi H, Parmely TJ, Sato S, Tomomori-Sato C, Banks CA, Kong SE,
            Szutorisz H, Swanson SK, Martin-Brown S, Washburn MP, Florens L, 
            Seidel CW, Lin C, Smith ER, Shilatifard A, Conaway RC, Conaway JW
  TITLE     Human mediator subunit MED26 functions as a docking site for 
            transcription elongation factors.
  JOURNAL   Cell. 2011 Jul 8;146(1):92-104.
  PUBMED    21729782
REFERENCE   2  (bases 1 to 6743)
  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: "Cell."; 
            date: "2011-07-8"; volume: "146(1)"; pages: "92-104"
COMMENT     Sequence Label: pcDNA5-F-AF9
FEATURES             Location/Qualifiers
     source          1..6743
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             907..930
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2695..2712)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2701..2925
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3058..3077)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3209..3256
                     /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             3264..4286
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3264..4286
                     /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    4416..4537
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4453..4472)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4507..4526
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4586..4602)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4586..4602)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4599..4621)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4610..4626
                     /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(4634..4640,4641..4658,4659..4664))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4679..4700
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4817..4834)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4988..5576)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5068..5087)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5747..6538,6539..6607))
                     /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     6370..6389
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6608..6712)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 atgccagact acaaggacga tgatgacaag ctcgagatgg ctagctcgtg tgccgtgcag
      961 gtgaagctgg agctggggca ccgcgcccag gtgaggaaaa aacccaccgt ggagggcttc
     1021 acccacgact ggatggtgtt cgtacgcggt ccggagcaca gtaacataca gcactttgtg
     1081 gagaaagtcg tcttccactt gcacgaaagc tttcctaggc caaaaagagt gtgcaaagat
     1141 ccaccttaca aagtagaaga atctgggtat gctggtttca ttttgccaat tgaagtttat
     1201 tttaaaaaca aggaagaacc taggaaagtc cgctttgatt atgacttatt cctgcatctt
     1261 gaaggccatc caccagtgaa tcacctccgc tgtgaaaagc taactttcaa caaccccaca
     1321 gaggacttta ggagaaagtt gctgaaggca ggaggggacc ctaataggag tattcatacc
     1381 agcagcagca gcagcagcag cagtagcagc agcagcagca gcagcagcag cagcagtagc
     1441 agcagcagca gcggcagcag cagcagcagt agcagcagca gtagcagcag cagcagcagc
     1501 agtagtacca gtttttcaaa gcctcacaaa ttaatgaagg agcacaagga aaaaccttct
     1561 aaagactcca gagaacataa aagtgccttc aaagaacctt ccagggatca caacaaatct
     1621 tccaaagaat cctctaagaa acccaaagaa aataaaccac tgaaagaaga gaaaatagtt
     1681 cctaagatgg ccttcaagga acctaaaccc atgtcaaaag agccaaaacc agatagtaac
     1741 ttactcacca tcaccagtgg acaagataag aaggctccta gtaaaaggcc gcccatttca
     1801 gattctgaag aactctcagc caaaaaaagg aaaaagagta gctcagaggc tttatttaaa
     1861 agtttttcta gcgcaccacc actgatactc acttgttctg ctgacaaaaa acagataaaa
     1921 gataaatctc atgtcaagat gggaaaggtc aaaattgaaa gtgagacatc agagaagaag
     1981 aaatcaacgt taccgccatt tgatgatatt gtggatccca atgattcaga tgtggaggag
     2041 aatatatcct ctaaatctga ttctgaacaa cccagtcctg ccagctccag ctccagctcc
     2101 agctccagct tcacaccatc ccagaccagg caacaaggtc ctttgaggtc tataatgaaa
     2161 gatctgcatt ctgatgacaa tgaggaggaa tcagatgaag tggaggataa cgacaatgac
     2221 tctgaaatgg agaggcctgt aaatagagga ggcagccgaa gtcgcagagt tagcttaagt
     2281 gatggcagcg atagtgaaag cagttctgct tcttcacccc tacatcacga acctccacca
     2341 cccttactaa aaaccaacaa caaccagatt cttgaagtga aaagtccaat aaagcaaagc
     2401 aaatcagata agcaaataaa gaatggtgaa tgtgacaagg catacctaga tgaactggta
     2461 gagcttcaca gaaggttaat gacattgaga gaaagacaca ttctgcagca gatcgtgaac
     2521 cttatagaag aaactggaca ctttcatatc acaaacacaa catttgattt tgatctttgc
     2581 tcgctggaca aaaccacagt ccgtaaacta cagagttacc tggaaacatc tggaacatcc
     2641 tgaggagcgg ccgctcgatc gagtctagag ggcccgttta aacccgctga tcagcctcga
     2701 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2761 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     2821 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     2881 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     2941 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3001 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3061 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3121 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3181 ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag
     3241 aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     3301 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     3361 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     3421 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     3481 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     3541 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     3601 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     3661 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     3721 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     3781 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     3841 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     3901 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     3961 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     4021 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     4081 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     4141 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     4201 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     4261 gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct
     4321 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4381 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4441 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4501 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     4561 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4621 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4681 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4741 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4801 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4861 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4921 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4981 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5041 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5101 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5161 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5221 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5281 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5341 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5401 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5461 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5521 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5581 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5641 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5701 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5761 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5821 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5881 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5941 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6001 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6061 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6121 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6181 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6241 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6301 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6361 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6421 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6481 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6541 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6601 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6661 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6721 cccgaaaagt gccacctgac gtc
//
