LOCUS       sequence-270456-        6259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  V5-His-tagged gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6259)
  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 6259)
  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-V5
FEATURES             Location/Qualifiers
     source          1..6259
                     /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"
     protein_bind    911..935
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1596..1620)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1637..1678
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1685..1726
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1757..1798
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1808..1825
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1848..1865)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1854..2078
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2124..2552
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2211..2230)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2421..2442
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2561..2581)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2566..2895
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2746..2881
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2808..2827
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2962..3756
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3016..3035)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3626..3645
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3932..4053
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3969..3988)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4023..4042
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4102..4118)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4102..4118)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4115..4137)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4126..4142
                     /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(4150..4156,4157..4174,4175..4180))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4195..4216
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4333..4350)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4504..5092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4584..4603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5263..6054,6055..6123))
                     /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     5886..5905
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6124..6228)
                     /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 gctggctagt
      901 taagctatca acaagtttgt acaaaaaagc aggctccacc atggaagcca tcgccaaata
      961 tgacttcaaa gctactgcag acgacgagct gagcttcaaa aggggggaca tcctcaaggt
     1021 tttgaacgaa gaatgtgatc agaactggta caaggcagag cttaatggaa aagacggctt
     1081 cattcccaag aactacatag aaatgaaacc acatccgtgg ttttttggca aaatccccag
     1141 agccaaggca gaagaaatgc ttagcaaaca gcggcacgat ggggcctttc ttatccgaga
     1201 gagtgagagc gctcctgggg acttctccct ctctgtcaag tttggaaacg atgtgcagca
     1261 cttcaaggtg ctccgagatg gagccgggaa gtacttcctc tgggtggtga agttcaattc
     1321 tttgaatgag ctggtggatt atcacagatc tacatctgtc tccagaaacc agcagatatt
     1381 cctgcgggac atagaacagg tgccacagca gccgacatac gtccaggccc tctttgactt
     1441 tgatccccag gaggatggag agctgggctt ccgccgggga gattttatcc atgtcatgga
     1501 taactcagac cccaactggt ggaaaggagc ttgccacggg cagaccggca tgtttccccg
     1561 caattatgtc acccccgtga accggaacgt cttggaccca gctttcttgt acaaagtggt
     1621 tgctctagaa ggctctggta aaccgattcc gaacccgctg ctgggtctgg attctaccgg
     1681 ttccggcaaa ccgatcccga acccgctgct gggcctggac tctaccggtt ctggtctaga
     1741 gggcccgcgg ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg
     1801 taccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
     1861 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1921 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1981 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2041 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2101 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2161 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2221 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2281 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2341 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2401 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2461 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2521 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2581 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2641 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2701 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     2761 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     2821 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     2881 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     2941 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3001 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3061 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3121 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3181 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3241 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3301 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3361 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3421 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3481 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3541 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3601 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3661 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3721 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgcgaaat
     3781 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     3841 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3901 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3961 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4021 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4081 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4141 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4201 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4261 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4321 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4381 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4441 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4501 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4561 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4621 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4681 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4741 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4801 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4861 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4921 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4981 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5041 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5101 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5161 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5221 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5281 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5341 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5401 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5461 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5521 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5581 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5641 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5701 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5761 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5821 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5881 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5941 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6001 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6061 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6121 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6181 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6241 aaaagtgcca cctgacgtc
//
