LOCUS       sequence-214589-        7481 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 7481)
  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 7481)
  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..7481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             350..607
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    654..779
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1272..1505
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1690..1734
                     /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    2029..2146
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2206..2509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2510..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             2906..2929
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3017..3073
                     /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(3203..3205,3206..3919)
                     /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(3245..3266)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3853..3874
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3996..4629
                     /label=5' LTR
                     /note="5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4678..4694)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4678..4694)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4691..4713)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4702..4718
                     /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(4726..4732,4733..4750,4751..4756))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4771..4792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4909..4926)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5069..5204
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     rep_origin      complement(5267..5855)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5347..5366)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6026..6817,6818..6886))
                     /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     6649..6668
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6887..6991)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7059..7077
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7115..7137)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7237..7256
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7450..7472
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7464..7481
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7465..7481
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaattgatat ccttgatctg tggatctacc acacacaagg ctacttccct gattagcaga
       61 actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc tacaagctag
      121 taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc agcttgttac
      181 accctgtgag cctgcatggg atggatgacc cggagagaga agtgttagag tggaggtttg
      241 acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac ttcaagaact
      301 gctgatatcg agcttgctac aagggacttt ccgctgggga ctttccaggg aggcgtggcc
      361 tgggcgggac tggggagtgg cgagccctca gatcctgcat ataagcagct gctttttgcc
      421 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
      481 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
      541 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
      601 tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc
      661 gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga
      721 gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca
      781 gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa
      841 agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag
      901 ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac
      961 catcccttca gacaggatca gaagaactta gatcattata taatacagta gcaaccctct
     1021 attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg
     1081 aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga
     1141 ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt
     1201 gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga
     1261 gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc
     1321 gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag
     1381 cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg
     1441 ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag
     1501 ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat
     1561 gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg
     1621 gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac
     1681 cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat
     1741 tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc
     1801 ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga
     1861 tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa
     1921 ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga
     1981 tctcgacggt atcggaccct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     2041 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     2101 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     2161 ggacagcaga aatccacttt gggtccgata agcttgggag ttccgcgtta cataacttac
     2221 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     2281 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     2341 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     2401 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     2461 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     2521 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     2581 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     2641 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     2701 tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
     2761 tgacctccat agaagacacc gggaccgatc cagcctccga attcgcaggt cgaccaccat
     2821 gaagcgcttc ctcttcctcc tactcaccat cagcctcctg gttatggtac agatacaaac
     2881 tggactctca ggacaaaacg acaccgacta caaagacgat gacgacaagt cagcatccag
     2941 caacataagc ggaggcattt tccttttctt cgtggccaat gccataatcc acctcttctg
     3001 cttcagtgga tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga
     3061 gaatcctgga ccggccaaac ctccggccaa ggcacaagtt gtgggatggc caccggtgag
     3121 atcataccgg aagaacgtga tggtttcctg ccaaaaatca agcggtggcc cggaggcggc
     3181 ggcgttcgtg aaagtagcta gcgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     3241 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtcc gcggcgaggg
     3301 cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     3361 gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcgtggcct gcttcagccg
     3421 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     3481 ccaggagcgc accatctctt tcaaggacga cggtacctac aagacccgcg ccgaggtgaa
     3541 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     3601 cggcaacatc ctggggcaca agctggagta caacttcaac agccacaacg tctatatcac
     3661 ggccgacaag cagaagaacg gcatcaaggc taacttcaag atccgccaca acgttgagga
     3721 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     3781 gctgctgccc gacaaccact acctgagcca tcagtccaag ctgagcaaag accccaacga
     3841 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatta cacatggcat
     3901 ggacgagctg tacaagtaag ctcgagtacc tttaagacca atgacttaca aggcagctgt
     3961 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     4021 aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca
     4081 gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct
     4141 agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt
     4201 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt
     4261 tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggagt acttcaagaa
     4321 ctgctgacat cgagcttgct acaagggact ttccgctggg gactttccag ggaggcgtgg
     4381 cctgggcggg actggggagt ggcgagccct cagatgctgc atataagcag ctgctttttg
     4441 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     4501 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     4561 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     4621 tctctagcag gatcctctag agtcgacctg caggcatgca agcttggcgt aatcatggtc
     4681 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4741 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4801 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4861 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4921 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4981 acggttatcc acagaatcag gggataacgc aggaaagaac atgcatctca attagtcagc
     5041 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     5101 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     5161 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctagatg tgagcaaaag
     5221 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5281 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5341 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5401 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5461 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5521 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5581 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5641 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5701 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5761 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5821 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5881 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5941 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6001 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6061 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6121 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6181 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6241 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6301 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6361 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6421 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6481 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6541 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6601 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6661 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6721 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6781 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6841 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6901 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6961 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7021 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     7081 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     7141 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     7201 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     7261 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     7321 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     7381 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     7441 cagggttttc ccagtcacga cgttgtaaaa cgacggccag t
//
