LOCUS       sequence-214605-        7546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing cell surface Flag-tag and monomeric GFP
            C-terminaly fused with auxin inducible degron.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7546)
  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 7546)
  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-mClover-smAID-P2A-CD5Flag2-bglpA
FEATURES             Location/Qualifiers
     source          1..7546
                     /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             join(2819..2821,2822..2824,2825..3535)
                     /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="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2864..2885)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3472..3493
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3671..3727
                     /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             3812..3835
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3916..3964
                     /note="synthetic polyadenylation signal"
     LTR             4061..4694
                     /label=5' LTR
                     /note="5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4743..4759)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4743..4759)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4756..4778)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4767..4783
                     /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(4791..4797,4798..4815,4816..4821))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4836..4857
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4974..4991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5134..5269
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     rep_origin      complement(5332..5920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5412..5431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6091..6882,6883..6951))
                     /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     6714..6733
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6952..7056)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7124..7142
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7180..7202)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7302..7321
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7515..7537
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7529..7546
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7530..7546
                     /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 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     2881 cgacgtaaac ggccacaagt tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg
     2941 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     3001 cgtgaccacc ttcggctacg gcgtggcctg cttcagccgc taccccgacc acatgaagca
     3061 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcttt
     3121 caaggacgac ggtacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     3181 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     3241 gctggagtac aacttcaaca gccacaacgt ctatatcacg gccgacaagc agaagaacgg
     3301 catcaaggct aacttcaaga tccgccacaa cgttgaggac ggcagcgtgc agctcgccga
     3361 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     3421 cctgagccat cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     3481 gctggagttc gtgaccgccg ccgggattac acatggcatg gacgagctgt acaagccagc
     3541 caaacctccg gccaaggcac aagttgtggg atggccaccg gtgagatcat accggaagaa
     3601 cgtgatggtt tcctgccaaa aatcaagcgg tggcccggag gcggcggcgt tcgtgaaagt
     3661 aggatccggc gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc
     3721 tggaccgaag cgcttcctct tcctcctact caccatcagc ctcctggtta tggtacagat
     3781 acaaactgga ctctcaggac aaaacgacac cgactacaaa gacgatgacg acaagtcagc
     3841 atccagcaac ataagcggag gcattttcct tttcttcgtg gccaatgcca taatccacct
     3901 cttctgcttc agttgaataa aatatcttta ttttcattac atctgtgtgt tggttttttg
     3961 tgtggtaccg catcttgctg aaaaactcga gtacctttaa gaccaatgac ttacaaggca
     4021 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     4081 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
     4141 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac
     4201 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc
     4261 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg
     4321 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggagtacttc
     4381 aagaactgct gacatcgagc ttgctacaag ggactttccg ctggggactt tccagggagg
     4441 cgtggcctgg gcgggactgg ggagtggcga gccctcagat gctgcatata agcagctgct
     4501 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     4561 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     4621 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     4681 gaaaatctct agcaggatcc tctagagtcg acctgcaggc atgcaagctt ggcgtaatca
     4741 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4801 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4861 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4921 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4981 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5041 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgca tctcaattag
     5101 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     5161 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     5221 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agatgtgagc
     5281 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5341 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5401 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5461 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5521 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5581 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5641 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5701 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5761 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5821 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5881 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5941 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6001 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6061 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6121 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6181 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6241 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6301 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6361 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6421 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6481 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6541 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6601 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6661 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6721 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6781 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6841 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6901 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6961 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7021 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7081 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     7141 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     7201 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     7261 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     7321 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     7381 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     7441 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     7501 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagt
//
