LOCUS       sequence-208138-        7490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing cell surface Flag-tag and monomeric GFP
            N-terminaly fused with auxin inducible degron.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7490)
  AUTHORS   Zotova A, Pichugin A, Atemasova A, Knyazhanskaya E, Lopatukhina E, 
            Mitkin N, Holmuhamedov E, Gottikh M, Kuprash D, Filatov A, Mazurov D
  TITLE     Isolation of gene-edited cells via knock-in of short 
            glycophosphatidylinositol-anchored epitope tags.
  JOURNAL   Sci Rep. 2019 Feb 28;9(1):3132. doi: 10.1038/s41598-019-40219-z.
  PUBMED    30816313
REFERENCE   2  (bases 1 to 7490)
  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: "Sci Rep."; 
            date: "2019-02-28"; volume: "9(1):3132. doi"; pages: " 
            10.1038/s41598-019-40219-z"
COMMENT     Sequence Label: pUCHR-CD5Flag2-P2A-smAID-mClover
FEATURES             Location/Qualifiers
     source          1..7490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             352..609
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    656..781
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1274..1507
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1692..1736
                     /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"
     misc_feature    2031..2148
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2208..2511
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2512..2713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2664..2684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2710..2734
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2914..2937
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3025..3081
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(3211..3213,3214..3927)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3253..3274)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3861..3882
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4004..4637
                     /label=5' LTR
                     /note="5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4686..4702)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4686..4702)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4699..4721)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4710..4726
                     /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)."
     promoter        complement(join(4734..4740,4741..4758,4759..4764))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4779..4800
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4917..4934)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5077..5212
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     rep_origin      complement(5276..5864)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5356..5375)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6035..6826,6827..6895))
                     /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     6658..6677
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6896..7000)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7068..7086
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7124..7146)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7246..7265
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7459..7481
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7473..7490
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7474..7490
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaattaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattagca
       61 gaactacaca ccagggccag gggtcagata tccactgacc tttggatggt gctacaagct
      121 agtaccagtt gagccagata aggtagaaga ggccaataaa ggagagaaca ccagcttgtt
      181 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtgttag agtggaggtt
      241 tgacagccgc ctagcatttc atcacgtggc ccgagagctg catccggagt acttcaagaa
      301 ctgctgatat cgagcttgct acaagggact ttccgctggg gactttccag ggaggcgtgg
      361 cctgggcggg actggggagt ggcgagccct cagatcctgc atataagcag ctgctttttg
      421 cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
      481 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
      541 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
      601 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
      661 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
      721 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
      781 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
      841 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
      901 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
      961 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     1021 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     1081 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg
     1141 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     1201 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     1261 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     1321 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     1381 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     1441 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     1501 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     1561 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     1621 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     1681 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     1741 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     1801 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     1861 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg
     1921 aaggaataga agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg
     1981 gatctcgacg gtatcggacc ctcgagacaa atggcagtat tcatccacaa ttttaaaaga
     2041 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     2101 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     2161 agggacagca gaaatccact ttgggtccga taagcttggg agttccgcgt tacataactt
     2221 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     2281 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     2341 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     2401 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg
     2461 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     2521 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacgggattt ccaagtctcc
     2581 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac ttccaaaatg
     2641 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     2701 tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
     2761 tgacctccat agaagacacc gggaccgatc cagcctccca agcttcgaat tcgcaggtcg
     2821 accaccatga agcgcttcct cttcctccta ctcaccatca gcctcctggt tatggtacag
     2881 atacaaactg gactctcagg acaaaacgac accgactaca aagacgatga cgacaagtca
     2941 gcatccagca acataagcgg aggcattttc cttttcttcg tggccaatgc cataatccac
     3001 ctcttctgct tcagtggatc cggcgcaaca aacttctctc tgctgaaaca agccggagat
     3061 gtcgaagaga atcctggacc ggccaaacct ccggccaagg cacaagttgt gggatggcca
     3121 ccggtgagat cataccggaa gaacgtgatg gtttcctgcc aaaaatcaag cggtggcccg
     3181 gaggcggcgg cgttcgtgaa agtagctagc gtgagcaagg gcgaggagct gttcaccggg
     3241 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtccgc
     3301 ggcgagggcg agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc
     3361 ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cgtggcctgc
     3421 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     3481 ggctacgtcc aggagcgcac catctctttc aaggacgacg gtacctacaa gacccgcgcc
     3541 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     3601 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaacgtc
     3661 tatatcacgg ccgacaagca gaagaacggc atcaaggcta acttcaagat ccgccacaac
     3721 gttgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     3781 ggccccgtgc tgctgcccga caaccactac ctgagccatc agtccaagct gagcaaagac
     3841 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggattaca
     3901 catggcatgg acgagctgta caagtaagct cgagtacctt taagaccaat gacttacaag
     3961 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     4021 tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct
     4081 gattggcaga actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc
     4141 tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc
     4201 cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag
     4261 tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca tccggagtac
     4321 ttcaagaact gctgacatcg agcttgctac aagggacttt ccgctgggga ctttccaggg
     4381 aggcgtggcc tgggcgggac tggggagtgg cgagccctca gatgctgcat ataagcagct
     4441 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     4501 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     4561 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     4621 gtggaaaatc tctagcagga tcctctagag tcgacctgca ggcatgcaag cttggcgtaa
     4681 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4741 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4801 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4861 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4921 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4981 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gcatctcaat
     5041 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     5101 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     5161 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagcatgt
     5221 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5281 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5341 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5401 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5461 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5521 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5581 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5641 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5701 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5761 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5821 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5881 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5941 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6001 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6061 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6121 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6181 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6241 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6301 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6361 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6421 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6481 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6541 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6601 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6661 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6721 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6781 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6841 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6901 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6961 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7021 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     7081 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     7141 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     7201 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     7261 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     7321 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     7381 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     7441 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
//
