LOCUS       Exported                6259 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  V5-His-tagged gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    GRB2-V5
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 May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6259
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    677..701
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1362..1386)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1403..1444
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1451..1492
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1523..1564
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1574..1591
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1614..1631)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1620..1844
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1890..2318
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1977..1996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2187..2208
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2327..2347)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2332..2661
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2512..2647
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2574..2593
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2728..3522
                     /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(2782..2801)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3392..3411
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3698..3819
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3735..3754)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3789..3808
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3868..3884)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3868..3884)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3881..3903)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3892..3908
                     /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(3916..3946)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3961..3982
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4099..4116)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4270..4858)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4350..4369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5029..5889)
                     /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     5652..5671
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5890..5994)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6069..6088)
                     /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 agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagttaag ctatcaacaa gtttgtacaa aaaagcaggc tccaccatgg aagccatcgc
      721 caaatatgac ttcaaagcta ctgcagacga cgagctgagc ttcaaaaggg gggacatcct
      781 caaggttttg aacgaagaat gtgatcagaa ctggtacaag gcagagctta atggaaaaga
      841 cggcttcatt cccaagaact acatagaaat gaaaccacat ccgtggtttt ttggcaaaat
      901 ccccagagcc aaggcagaag aaatgcttag caaacagcgg cacgatgggg cctttcttat
      961 ccgagagagt gagagcgctc ctggggactt ctccctctct gtcaagtttg gaaacgatgt
     1021 gcagcacttc aaggtgctcc gagatggagc cgggaagtac ttcctctggg tggtgaagtt
     1081 caattctttg aatgagctgg tggattatca cagatctaca tctgtctcca gaaaccagca
     1141 gatattcctg cgggacatag aacaggtgcc acagcagccg acatacgtcc aggccctctt
     1201 tgactttgat ccccaggagg atggagagct gggcttccgc cggggagatt ttatccatgt
     1261 catggataac tcagacccca actggtggaa aggagcttgc cacgggcaga ccggcatgtt
     1321 tccccgcaat tatgtcaccc ccgtgaaccg gaacgtcttg gacccagctt tcttgtacaa
     1381 agtggttgct ctagaaggct ctggtaaacc gattccgaac ccgctgctgg gtctggattc
     1441 taccggttcc ggcaaaccga tcccgaaccc gctgctgggc ctggactcta ccggttctgg
     1501 tctagagggc ccgcggttcg aaggtaagcc tatccctaac cctctcctcg gtctcgattc
     1561 tacgcgtacc ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac
     1621 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1681 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1741 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1801 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1861 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1921 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1981 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2041 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2101 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2161 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2221 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2281 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     2341 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2401 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2461 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2521 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2581 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     2641 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     2701 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     2761 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     2821 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     2881 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     2941 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3001 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3061 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3121 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3181 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3241 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     3301 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     3361 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     3421 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3481 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3541 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     3601 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3661 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     3721 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3781 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     3841 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3901 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3961 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4021 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4081 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4141 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4201 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4261 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4321 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4381 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4441 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4501 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4561 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4621 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4681 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4741 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4801 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4861 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4921 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4981 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5041 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5101 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5161 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5221 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5281 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5341 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5401 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5461 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5521 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5581 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5641 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5701 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5761 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5821 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5881 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5941 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6001 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     6061 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     6121 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     6181 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     6241 cgggccagat atacgcgtt
//
