LOCUS       sequence-297074-        6012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  StrepIII-HA-tagged gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6012)
  AUTHORS   Yadav L, Pietila E, Ohman T, Liu X, Mahato AK, Sidorova Y, Lehti K, 
            Saarma M, Varjosalo M
  TITLE     PTPRA Phosphatase Regulates GDNF-Dependent RET Signaling and 
            Inhibits the RET Mutant MEN2A Oncogenic Potential.
  JOURNAL   iScience. 2020 Feb 21;23(2):100871. doi: 10.1016/j.isci.2020.100871.
            Epub 2020 Jan 31.
  PUBMED    32062451
REFERENCE   2  (bases 1 to 6012)
  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: "iScience.";
            date: "2020-02-21"; volume: "23(2):100871. doi"; pages: " 
            10.1016/j.isci.2020.100871. Epub 2020 Jan 31"
COMMENT     Sequence Label: GRB2-SHN
FEATURES             Location/Qualifiers
     source          1..6012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             830..856
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             887..910
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             947..970
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    983..1007
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1668..1692)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1730..1747)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1736..1960
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2093..2112)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2244..2291
                     /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             2299..3321
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2299..3321
                     /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    3451..3572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3488..3507)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3542..3561
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3621..3637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3621..3637)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3634..3656)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3645..3661
                     /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(3669..3675,3676..3693,3694..3699))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3714..3735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3852..3869)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4023..4611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4103..4122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4782..5573,5574..5642))
                     /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     5405..5424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5643..5747)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5822..5841)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccact agtccagtgt
      781 ggtggaattc tgcagatatc cagcacagtg gcggccgctc gagaccatgt acccatacga
      841 tgttcccgac tacgccggta ccgagctcgg atccaccatg gctagctgga gccacccgca
      901 gttcgagaaa ggtggaggtt ccggaggtgg atcgggaggt ggatcgtgga gccacccgca
      961 gttcgaaaaa gcggccgata tcacaagttt gtacaaaaaa gcaggctcca ccatggaagc
     1021 catcgccaaa tatgacttca aagctactgc agacgacgag ctgagcttca aaagggggga
     1081 catcctcaag gttttgaacg aagaatgtga tcagaactgg tacaaggcag agcttaatgg
     1141 aaaagacggc ttcattccca agaactacat agaaatgaaa ccacatccgt ggttttttgg
     1201 caaaatcccc agagccaagg cagaagaaat gcttagcaaa cagcggcacg atggggcctt
     1261 tcttatccga gagagtgaga gcgctcctgg ggacttctcc ctctctgtca agtttggaaa
     1321 cgatgtgcag cacttcaagg tgctccgaga tggagccggg aagtacttcc tctgggtggt
     1381 gaagttcaat tctttgaatg agctggtgga ttatcacaga tctacatctg tctccagaaa
     1441 ccagcagata ttcctgcggg acatagaaca ggtgccacag cagccgacat acgtccaggc
     1501 cctctttgac tttgatcccc aggaggatgg agagctgggc ttccgccggg gagattttat
     1561 ccatgtcatg gataactcag accccaactg gtggaaagga gcttgccacg ggcagaccgg
     1621 catgtttccc cgcaattatg tcacccccgt gaaccggaac gtcttggacc cagctttctt
     1681 gtacaaagtg gtgacgtaag ctaggggccc gtttaaaccc gctgatcagc ctcgactgtg
     1741 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1801 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1861 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1921 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     1981 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2041 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2101 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2161 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2221 tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt
     2281 ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     2341 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     2401 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     2461 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     2521 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     2581 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     2641 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     2701 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     2761 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     2821 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     2881 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     2941 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     3001 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     3061 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     3121 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     3181 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     3241 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     3301 cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat
     3361 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3421 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     3481 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3541 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     3601 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3661 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3721 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3781 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3841 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3901 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3961 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4021 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4081 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4141 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4201 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4261 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4321 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4381 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4441 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4501 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4561 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4621 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4681 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4741 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4801 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4861 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4921 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4981 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5041 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5101 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5161 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5221 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5281 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5341 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5401 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5461 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5521 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5581 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5641 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5701 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5761 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     5821 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     5881 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     5941 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     6001 gatatacgcg tt
//
