LOCUS       sequence-424716-        6405 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of a gene, accompanied by the expression of the 
            affinity sorting tag GST-EGFP-GPIDAF driven by an SV40 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6405)
  AUTHORS   Ma S, Yang L, Zuo Q, Huang Q
  TITLE     GPI-anchored glutathione S-transferase as marker allows affinity 
            sorting of transfection-positive cells.
  JOURNAL   Front Mol Biosci. 2022 Sep 29;9:1016090. doi: 
            10.3389/fmolb.2022.1016090. eCollection 2022.
  PUBMED    36250010
REFERENCE   2  (bases 1 to 6405)
  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: "Front Mol 
            Biosci."; date: "2022-09-29"; volume: "9:1016090. doi"; pages: " 
            10.3389/fmolb.2022.1016090. eCollection 2022"
COMMENT     Sequence Label: pcDNA3.1-GST-EGFP-GPIDAF
FEATURES             Location/Qualifiers
     source          1..6405
                     /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"
     CDS             1000..1041
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1051..1068
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1091..1108)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1097..1321
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1367..1795
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1454..1473)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1664..1685
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1804..1824)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1809..2138
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1989..2124
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2051..2070
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2328..2984
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2942..2964
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2991..3008
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      3015..3024
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3021..3023,3024..3026,3027..3737)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3021..3023,3024..3026,3027..3737)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3066..3087)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3327..3346)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3674..3695
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4078..4199
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4115..4134)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4169..4188
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4248..4264)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4248..4264)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4261..4283)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4272..4288
                     /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(4296..4302,4303..4320,4321..4326))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4341..4362
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4479..4496)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4650..5238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4730..4749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5409..6200,6201..6269))
                     /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     6032..6051
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6270..6374)
                     /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 taagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca
      961 gcacagtggc ggccgctcga gtctagaggg cccttcgaag gtaagcctat ccctaaccct
     1021 ctcctcggtc tcgattctac gcgtaccggt catcatcacc atcaccattg agtttaaacc
     1081 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     1141 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     1201 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     1261 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     1321 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     1381 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1441 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1501 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1561 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1621 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1681 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1741 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     1801 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     1861 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     1921 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     1981 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2041 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     2101 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     2161 ccattttcgg atctgatcaa gagacaggat gaggatcgtt taccatgacc gtggctagac
     2221 ccagcgtgcc tgctgccctg cctctgctgg gagagctgcc tagactgctg ctcctggtgc
     2281 tcctgtgcct gcctgctgtg tggggaagcg cctggagcca ccctcagatg ggctccccta
     2341 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
     2401 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
     2461 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
     2521 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
     2581 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
     2641 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
     2701 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
     2761 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
     2821 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
     2881 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
     2941 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggttccgc
     3001 gtggatcccc ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     3061 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     3121 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     3181 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     3241 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     3301 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     3361 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     3421 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     3481 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     3541 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     3601 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     3661 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     3721 tggacgagct gtacaagtcc ggccggactc agatcagccg gaccaccaag cacttccacg
     3781 agaccacccc taacaagggc agcggcacca ccagcggcac cacccggctg ctgagcggcc
     3841 acacctgctt caccctgacc ggcctgctgg gcaccctggt gaccatgggc ctgctgacct
     3901 aggcgggact ctggggttcg cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     3961 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4021 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     4081 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4141 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4201 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4261 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4321 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4381 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4441 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4501 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4561 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4621 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4681 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4741 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4801 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4861 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4921 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4981 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5041 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5101 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5161 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5221 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5281 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5341 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5401 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5461 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5521 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5581 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5641 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5701 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5761 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5821 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5881 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5941 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6001 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6061 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6121 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6181 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6241 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6301 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6361 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
