LOCUS       sequence-429328-        5730 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-GPIBY55 under a 
            CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5730)
  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 5730)
  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-GPIBY55
FEATURES             Location/Qualifiers
     source          1..5730
                     /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             1140..1796
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1754..1776
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1803..1820
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      1827..1836
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1833..1835,1836..1838,1839..2549)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1833..1835,1836..1838,1839..2549)
                     /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(1878..1899)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2139..2158)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2486..2507
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2679..2735
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2859..2980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2896..2915)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2950..2969
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   2993..3133
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2993..3122
                     /label=AAV2 ITR
     rep_origin      3208..3663
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3295..3314)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3505..3526
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3680..3699)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3799..3821
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3859..3877)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3945..4049
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4050..4118,4119..4910)
                     /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(4268..4287)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5081..5669
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5570..5589
                     /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 accatgctgc tggagcctgg cagaggctgc tgcgctctgg
     1081 ccatcctgct ggccatcgtg gacatccaga gcggaggcag cgcctggagc caccctcaga
     1141 tgggctcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc actcgacttc
     1201 ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat gaaggtgata
     1261 aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct tattatattg
     1321 atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct gacaagcaca
     1381 acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa ggagcggttt
     1441 tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa actctcaaag
     1501 ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt ttatgtcata
     1561 aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat gacgctcttg
     1621 atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta gtttgtttta
     1681 aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc aagtatatag
     1741 catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct ccaaaatcgg
     1801 atctggttcc gcgtggatcc ccggtcgcca ccatggtgag caagggcgag gagctgttca
     1861 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1921 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1981 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     2041 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2101 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2161 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2221 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2281 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     2341 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2401 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     2461 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2521 tcactctcgg catggacgag ctgtacaagt ccggccggac tcagatccgg cagcacctgg
     2581 agttcagcca caacgagggc accctgagca gcggcttcct ccaggagaag gtgtgggtga
     2641 tgctggtgac cagcctggtg gctctccagg ccctgtagag atctcgagct caagcttcga
     2701 attctgcagt cgacggtacc gcgggcccgg gatccaccgg atctagataa ctgatcataa
     2761 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     2821 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     2881 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     2941 attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtggcc gcaggaaccc
     3001 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     3061 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     3121 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3181 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     3241 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     3301 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3361 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3421 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3481 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     3541 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     3601 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3661 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     3721 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3781 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3841 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     3901 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3961 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4021 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4081 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4141 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4201 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4261 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4321 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4381 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4441 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4501 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     4561 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     4621 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     4681 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     4741 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     4801 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4861 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4921 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4981 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5041 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5101 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5161 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5221 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5281 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5341 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5401 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5461 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5521 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5581 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5641 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5701 tggccttttg ctggcctttt gctcacatgt
//
