LOCUS       sequence-429329-        5793 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a single-guide RNA under a U6 promoter, accompanied by 
            the expression of the affinity sorting tag GST-EGFP-GPIDAF under a 
            CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5793)
  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 5793)
  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: psgRNA-GST-EGFP-GPIDAF
FEATURES             Location/Qualifiers
     source          1..5793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        496..799
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        800..1003
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     954..974
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1167..1823
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1781..1803
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1830..1847
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      1854..1863
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1860..1862,1863..1865,1866..2576)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1860..1862,1863..1865,1866..2576)
                     /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(1905..1926)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2166..2185)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2513..2534
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2742..2798
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2922..3043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2959..2978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3013..3032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3056..3196
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3056..3185
                     /label=AAV2 ITR
     rep_origin      3271..3726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3358..3377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3568..3589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3743..3762)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3862..3884
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3922..3940)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4008..4112
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4113..4181,4182..4973)
                     /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     complement(4331..4350)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5144..5732
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5633..5652
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtactt attaatagta atcaattacg gggtcattag ttcatagccc
      481 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      541 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      601 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      661 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      721 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      781 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      841 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      901 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      961 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
     1021 gatccgctag cgctaccggt accatgaccg tggctagacc cagcgtgcct gctgccctgc
     1081 ctctgctggg agagctgcct agactgctgc tcctggtgct cctgtgcctg cctgctgtgt
     1141 ggggaagcgc ctggagccac cctcagatgg gctcccctat actaggttat tggaaaatta
     1201 agggccttgt gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc
     1261 atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg
     1321 agtttcccaa tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca
     1381 tcatacgtta tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag
     1441 agatttcaat gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat
     1501 atagtaaaga ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga
     1561 aaatgttcga agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc
     1621 ctgacttcat gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg
     1681 atgcgttccc aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata
     1741 agtacttgaa atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg
     1801 gtggtggcga ccatcctcca aaatcggatc tggttccgcg tggatccccg gtcgccacca
     1861 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1921 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1981 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     2041 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     2101 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2161 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2221 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2281 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     2341 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2401 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2461 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2521 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg
     2581 gccggactca gatcagccgg accaccaagc acttccacga gaccacccct aacaagggca
     2641 gcggcaccac cagcggcacc acccggctgc tgagcggcca cacctgcttc accctgaccg
     2701 gcctgctggg caccctggtg accatgggcc tgctgaccta gagatctcga gctcaagctt
     2761 cgaattctgc agtcgacggt accgcgggcc cgggatccac cggatctaga taactgatca
     2821 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     2881 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     2941 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3001 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgtg gccgcaggaa
     3061 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     3121 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     3181 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3241 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     3301 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     3361 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3421 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3481 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3541 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3601 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     3661 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     3721 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3781 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3841 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3901 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     3961 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4021 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4081 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4141 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4201 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4261 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4321 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4381 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     4441 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     4501 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     4561 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     4621 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     4681 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     4741 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     4801 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     4861 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     4921 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     4981 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5041 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5101 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5161 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5221 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5281 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5341 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5401 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5461 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5521 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5581 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5641 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5701 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5761 tcctggcctt ttgctggcct tttgctcaca tgt
//
