LOCUS       sequence-203336-        5216 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of your protein of interest in fusion with HA at the 
            N-terminus. The tag is cleavable by TEV protease..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5216)
  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 5216)
  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: "PLoS One.";
            date: "2018-03-28"; volume: "13(3):e0194887. doi"; pages: " 
            10.1371/journal.pone.0194887. eCollection 2018"
COMMENT     Sequence Label: pKK-HA-TEV
FEATURES             Location/Qualifiers
     source          1..5216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        13..392
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        393..596
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     547..567
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    598..616
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    619..637
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     647..671
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             771..797
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             804..824
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             865..885
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(946..963)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    952..1176
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1309..1328)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1460..1507
                     /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             1515..2537
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1515..2537
                     /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    2667..2788
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2704..2723)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2758..2777
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2837..2853)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2837..2853)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2850..2872)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2861..2877
                     /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(2885..2891,2892..2909,2910..2915))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2930..2951
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3068..3085)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3239..3827)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3319..3338)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3998..4789,4790..4858))
                     /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     4621..4640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4859..4963)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5038..5057)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
       61 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      121 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      181 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      241 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      301 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      361 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      421 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      481 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      541 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc
      601 ctatcagtga tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga
      661 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg
      721 accgatccag cctccggact ctggcgttta aacttaagct tggtaccatg tacccctacg
      781 acgtgcccga ctacgctgga tccgaaaacc tgtacttcca aggaaccggt ggtcacctgt
      841 acaatcgatc ctgcaggagc tagcgagaat ttgtattttc agggtgatat ccagcacagt
      901 ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
      961 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     1021 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     1081 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     1141 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     1201 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1261 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1321 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1381 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1441 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     1501 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     1561 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     1621 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     1681 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     1741 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     1801 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     1861 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     1921 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     1981 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     2041 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     2101 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     2161 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     2221 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     2281 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     2341 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     2401 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     2461 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     2521 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     2581 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     2641 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     2701 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2761 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     2821 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2881 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2941 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3001 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3061 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3121 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3181 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3241 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3301 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3361 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3421 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3481 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3541 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3601 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3661 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3721 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3781 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3841 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3901 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3961 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4021 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4081 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4141 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4201 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4261 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4321 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4381 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4441 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4501 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4561 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4621 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4681 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4741 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4801 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4861 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4921 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4981 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     5041 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     5101 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     5161 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggcca
//
