LOCUS       sequence-422941-        7480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For lentiviral expression of shRNA under a U6 promoter, accompanied 
            by the expression of the affinity sorting tag TST-EGFP-GPIDAF driven
            by an hPGK promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7480)
  AUTHORS   Yang L, Cui L, Ma S, Zuo Q, Huang Q
  TITLE     A Gene Transfer-Positive Cell Sorting System Utilizing 
            Membrane-Anchoring Affinity Tag.
  JOURNAL   Front Bioeng Biotechnol. 2022 Jun 16;10:930966. doi: 
            10.3389/fbioe.2022.930966. eCollection 2022.
  PUBMED    35782508
REFERENCE   2  (bases 1 to 7480)
  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 
            Bioeng Biotechnol."; date: "2022-06-16"; volume: "10:930966. doi"; 
            pages: " 10.3389/fbioe.2022.930966. eCollection 2022"
COMMENT     Sequence Label: pLKO.1-TST-EGFP-GPIDAF
FEATURES             Location/Qualifiers
     source          1..7480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..511
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     430..448
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(635..640,641..664,665..700,701..724)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     regulatory      731..740
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(737..739,740..742,743..1453)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(737..739,740..742,743..1453)
                     /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(782..803)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1043..1062)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1390..1411
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             1690..1923
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1995..2116
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2032..2051)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2086..2105
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2156..2291
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2311..2330)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2312..2330)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2340..2357)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2340..2356)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2349..2371)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2498..2953
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2585..2604)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2795..2816
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2979..3083
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3084..3152,3153..3944)
                     /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(3302..3321)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4115..4703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4604..4623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4857..4874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4991..5012
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5027..5032,5033..5050,5051..5057)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5065..5081
                     /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)."
     primer_bind     5070..5092
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5089..5105
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5089..5105
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5124..5144
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5126..5144
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5172..5398
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5399..5579
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5626..5751
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6244..6477
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6662..6706
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        7004..7252
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7004..7024
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7175..7194
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    7315..7432
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc
       61 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc
      121 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc
      181 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac
      241 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc
      301 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag
      361 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct
      421 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct
      481 cgttgaccga atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccgt
      541 ggctagaccc agcgtgcctg ctgccctgcc tctgctggga gagctgccta gactgctgct
      601 cctggtgctc ctgtgcctgc ctgctgtgtg gggaagcgcc tggagccacc ctcagttcga
      661 gaaaggagga ggcagcggcg gcggaagcgg aggctccgct tggtcccacc cacagttcga
      721 aaagccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      781 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      841 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      901 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      961 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1021 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1081 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1141 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1201 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1261 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1321 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1381 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1441 cgagctgtac aagtccggcc ggactcagat cagccggacc accaagcact tccacgagac
     1501 cacccctaac aagggcagcg gcaccaccag cggcaccacc cggctgctga gcggccacac
     1561 ctgcttcacc ctgaccggcc tgctgggcac cctggtgacc atgggcctgc tgacctgagg
     1621 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     1681 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
     1741 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     1801 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     1861 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     1921 gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     1981 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     2041 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     2101 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     2161 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2221 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     2281 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     2341 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     2401 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     2461 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     2521 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2581 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2641 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2701 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2761 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2821 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2881 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2941 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3001 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3061 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3121 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3181 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3241 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3301 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3361 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3421 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3481 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3541 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3601 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3661 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3721 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3781 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3841 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3901 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3961 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4021 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4081 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4141 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4201 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4261 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4321 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4381 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4441 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4501 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4561 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4621 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4681 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4741 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4801 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4861 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     4921 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4981 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     5041 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     5101 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
     5161 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
     5221 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag
     5281 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac
     5341 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg
     5401 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     5461 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     5521 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     5581 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
     5641 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
     5701 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
     5761 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     5821 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     5881 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     5941 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     6001 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     6061 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     6121 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     6181 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     6241 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     6301 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     6361 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     6421 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     6481 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     6541 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     6601 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     6661 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     6721 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     6781 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     6841 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     6901 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg
     6961 gtatcgatca cgagactagc ctcgagcggc cgcccccttc accgagggcc tatttcccat
     7021 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     7081 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     7141 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     7201 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggtccgca
     7261 ggtatgcacg cgtgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa
     7321 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     7381 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     7441 ttacagggac agcagagatc cactttggcc gcggctcgag
//
