LOCUS       sequence-422939-        7414 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For lentiviral expression of shRNA under a U6 promoter, accompanied 
            by the expression of the affinity sorting tag TST-EGFP-GPIBY55 
            driven by an hPGK promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7414)
  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 7414)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-GPIBY55
FEATURES             Location/Qualifiers
     source          1..7414
                     /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(605..610,611..634,635..670,671..694)
                     /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      701..710
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(707..709,710..712,713..1423)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(707..709,710..712,713..1423)
                     /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(752..773)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1013..1032)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1360..1381
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             1624..1857
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1929..2050
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1966..1985)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2020..2039
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2090..2225
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2245..2264)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2246..2264)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2274..2291)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2274..2290)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2283..2305)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2432..2887
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2519..2538)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2729..2750
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2913..3017
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3018..3086,3087..3878)
                     /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(3236..3255)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4049..4637
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4538..4557
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4791..4808
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4925..4946
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4961..4966,4967..4984,4985..4991)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4999..5015
                     /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     5004..5026
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5023..5039
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5023..5039
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5058..5078
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5060..5078
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5106..5332
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5333..5513
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5560..5685
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6178..6411
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6596..6640
                     /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        6938..7186
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6938..6958
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7109..7128
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    7249..7366
                     /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 gggggatccg gagcttacca tgctgctgga
      541 gcctggcaga ggctgctgcg ctctggccat cctgctggcc atcgtggaca tccagagcgg
      601 aggcagcgcc tggagccacc ctcagttcga gaaaggagga ggcagcggcg gcggaagcgg
      661 aggctccgct tggtcccacc cacagttcga aaagccggtc gccaccatgg tgagcaaggg
      721 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      781 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      841 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
      901 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
      961 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1021 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1081 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1141 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1201 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1261 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1321 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1381 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggcc ggactcagat
     1441 ccggcagcac ctggagttca gccacaacga gggcaccctg agcagcggct tcctccagga
     1501 gaaggtgtgg gtgatgctgg tgaccagcct ggtggctctc caggccctgt gaggtacctt
     1561 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     1621 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     1681 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     1741 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     1801 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     1861 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     1921 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     1981 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     2041 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     2101 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     2161 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     2221 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     2281 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     2341 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     2401 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     2461 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2521 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2581 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2641 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2701 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2761 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2821 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     2881 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2941 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3001 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3061 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3121 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3181 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3241 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     3301 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3361 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3421 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3481 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3541 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3601 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3661 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3721 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3781 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3841 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3901 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3961 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4021 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4081 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4141 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4201 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4261 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4321 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4381 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4441 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4501 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4561 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4621 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4681 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4741 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4801 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     4861 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     4921 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     4981 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     5041 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag
     5101 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
     5161 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
     5221 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
     5281 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
     5341 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     5401 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     5461 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
     5521 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
     5581 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
     5641 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
     5701 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
     5761 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
     5821 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
     5881 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
     5941 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
     6001 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
     6061 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     6121 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     6181 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     6241 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     6301 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     6361 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     6421 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     6481 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     6541 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     6601 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     6661 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     6721 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     6781 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     6841 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     6901 atcacgagac tagcctcgag cggccgcccc cttcaccgag ggcctatttc ccatgattcc
     6961 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     7021 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     7081 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     7141 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggtc cgcaggtatg
     7201 cacgcgtgaa ttctcgacct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     7261 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     7321 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     7381 ggacagcaga gatccacttt ggccgcggct cgag
//
