LOCUS       sequence-429326-        5778 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-GPICEAM7 under a
            CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5778)
  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 5778)
  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-GPICEAM7
FEATURES             Location/Qualifiers
     source          1..5778
                     /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             1173..1829
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1787..1809
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1836..1853
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      1860..1869
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1866..1868,1869..1871,1872..2582)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1866..1868,1869..1871,1872..2582)
                     /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(1911..1932)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2172..2191)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2519..2540
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2727..2783
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2907..3028
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2944..2963)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2998..3017
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3041..3181
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3041..3170
                     /label=AAV2 ITR
     rep_origin      3256..3711
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3343..3362)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3553..3574
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3728..3747)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3847..3869
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3907..3925)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3993..4097
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4098..4166,4167..4958)
                     /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(4316..4335)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5129..5717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5618..5637
                     /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 accatgggca gccctagcgc ctgcccttac agagtgtgca
     1081 tcccctggca gggcctgctg ctgaccgcca gcctgctgac cttctggaac ctgcccaaca
     1141 gcgcccagac cagcgcctgg agccaccctc agatgggctc ccctatacta ggttattgga
     1201 aaattaaggg ccttgtgcaa cccactcgac ttcttttgga atatcttgaa gaaaaatatg
     1261 aagagcattt gtatgagcgc gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg
     1321 gtttggagtt tcccaatctt ccttattata ttgatggtga tgttaaatta acacagtcta
     1381 tggccatcat acgttatata gctgacaagc acaacatgtt gggtggttgt ccaaaagagc
     1441 gtgcagagat ttcaatgctt gaaggagcgg ttttggatat tagatacggt gtttcgagaa
     1501 ttgcatatag taaagacttt gaaactctca aagttgattt tcttagcaag ctacctgaaa
     1561 tgctgaaaat gttcgaagat cgtttatgtc ataaaacata tttaaatggt gatcatgtaa
     1621 cccatcctga cttcatgttg tatgacgctc ttgatgttgt tttatacatg gacccaatgt
     1681 gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa
     1741 ttgataagta cttgaaatcc agcaagtata tagcatggcc tttgcagggc tggcaagcca
     1801 cgtttggtgg tggcgaccat cctccaaaat cggatctggt tccgcgtgga tccccggtcg
     1861 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1921 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1981 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2041 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2101 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2161 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2221 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2281 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2341 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2401 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2461 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2521 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2581 agtccggccg gactcagatc agccggagcg accctgtgac cctgaacgtg agatacgaga
     2641 gcgtgcaggc cagcagccct gacctgagcg ccggcaccgc cgtgagcatc atgatcggcg
     2701 tgctggctgg catggctctg atctagagat ctcgagctca agcttcgaat tctgcagtcg
     2761 acggtaccgc gggcccggga tccaccggat ctagataact gatcataatc agccatacca
     2821 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     2881 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     2941 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3001 gtttgtccaa actcatcaat gtatcttaac gcgtggccgc aggaacccct agtgatggag
     3061 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3121 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     3181 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     3241 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3301 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     3361 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3421 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     3481 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3541 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     3601 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     3661 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     3721 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     3781 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3841 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     3901 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3961 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4021 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4081 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4141 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4201 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4261 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4321 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4381 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4441 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4501 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4561 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4621 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4681 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4741 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4801 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4861 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4921 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4981 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5041 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5101 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5161 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5221 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5281 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5341 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5401 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5461 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5521 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5581 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5641 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5701 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5761 ggccttttgc tcacatgt
//
