LOCUS       sequence-203379-        5589 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of your protein of interest in fusion with two IgG 
            binding domains of Staphylococcus aureus protein A at the C-terminus
            (cleavable by TEV) (PMID: 3507693, 10504710). Protein purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5589)
  AUTHORS   Szczesny RJ, Kowalska K, Klosowska-Kosicka K, Chlebowski A, Owczarek
            EP, Warkocki Z, Kulinski TM, Adamska D, Affek K, Jedroszkowiak A, 
            Kotrys AV, Tomecki R, Krawczyk PS, Borowski LS, Dziembowski A
  TITLE     Versatile approach for functional analysis of human proteins and 
            efficient stable cell line generation using FLP-mediated 
            recombination system.
  JOURNAL   PLoS One. 2018 Mar 28;13(3):e0194887. doi: 
            10.1371/journal.pone.0194887. eCollection 2018.
  PUBMED    29590189
REFERENCE   2  (bases 1 to 5589)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2018-03-28"; volume: "13(3):e0194887. doi"; pages: " 
            10.1371/journal.pone.0194887. eCollection 2018"
COMMENT     Sequence Label: pKK-TEV-ProteinA
FEATURES             Location/Qualifiers
     source          1..5589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..58)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        230..609
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        610..813
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     764..784
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    815..833
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    836..854
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     864..888
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             997..1017
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1058..1078
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1118..1291
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             1292..1465
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNGAQAPK"
     primer_bind     complement(1536..1553)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1542..1766
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1899..1918)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2050..2097
                     /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             2105..3127
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2105..3127
                     /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    3257..3378
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3294..3313)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3348..3367
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3427..3443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3427..3443)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3440..3462)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3451..3467
                     /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(3475..3481,3482..3499,3500..3505))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3520..3541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3658..3675)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3829..4417)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3909..3928)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4588..5379,5380..5448))
                     /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     5211..5230
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5449..5553)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
       61 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
      121 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
      181 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      241 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      301 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      361 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      421 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      481 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      541 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      601 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      661 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      721 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      781 cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag agatctccct
      841 atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc
      901 catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct ccggactctg
      961 gcgtttaaac ttaagcttgg taccgagctc ggatccgaaa acctgtactt ccaaggaacc
     1021 ggtggtcacc tgtacaatcg atcctgcagg agctagcgag aatttgtatt ttcagggtga
     1081 tatcaaaacc gcggctcttg cgcaacacga tgaagccgtg gacaacaaat tcaacaaaga
     1141 acaacaaaac gcgttctatg agatcttaca tttacctaac ttaaacgaag aacaacgaaa
     1201 cgccttcatc caaagtttaa aagatgaccc aagccaaagc gctaaccttt tagcagaagc
     1261 taaaaagcta aatgatgctc aggcgccgaa agtagacaac aaattcaaca aagaacaaca
     1321 aaacgcgttc tatgagatct tacatttacc taacttaaac gaagaacaac gaaacgcctt
     1381 catccaaagt ttaaaagatg acccaagcca aagcgctaac cttttagcag aagctaaaaa
     1441 gctaaatggt gctcaggcgc cgaaagtaga cgcgaattcc gcggggaagt caacctgact
     1501 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     1561 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1621 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     1681 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     1741 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     1801 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1861 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1921 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1981 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2041 acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt
     2101 ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     2161 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     2221 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     2281 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     2341 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     2401 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
     2461 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     2521 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     2581 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     2641 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     2701 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     2761 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     2821 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     2881 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     2941 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     3001 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     3061 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     3121 ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     3181 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     3241 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     3301 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3361 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     3421 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3481 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3541 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3601 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3661 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3721 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3781 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3841 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3901 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3961 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4021 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4081 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4141 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4201 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4261 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4321 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4381 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4441 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4501 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4561 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4621 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4681 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4741 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4801 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4861 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4921 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4981 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5041 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5101 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5161 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5221 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5281 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5341 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5401 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5461 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5521 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5581 cgtcgacgg
//
