LOCUS       Exported                8301 bp DNA     circular SYN 11-SEP-2025
DEFINITION  Exported.
ACCESSION   V012838
VERSION     .
KEYWORDS    pLKO.1-EGFP-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8301)
  AUTHORS   hfytfiu
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8301)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 11, 2025 from SnapGene 6.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          join(2176..8301,1..2175)
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             102..146
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
     CDS             295..336
                     /label=Protein Tat
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
     promoter        444..684
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     enhancer        706..1009
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1010..1213
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      1242..1251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1248..1967
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    2006..2123
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        2172..2682
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     promoter        2172..2175
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             2704..3303
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     LTR             3431..3664
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3736..3870
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      3897..4032
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(4053..4071)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4081..4097)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      4239..4694
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4720..4824
                     /label=AmpR promoter
     CDS             4825..5685
                     /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"
     rep_origin      5856..6444
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    6732..6753
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        6768..6798
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6806..6822
                     /label=lac operator
                     /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     6830..6846
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6867..6885
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6913..7139
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7140..7320
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7367..7492
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    7985..8218
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
       61 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
      121 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
      181 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
      241 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
      301 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag
      361 acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc
      421 ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata
      481 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
      541 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      601 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      661 ctttatatat cttgtggaaa ggacgaaaca ccggtcggaa ttccgcgtta cataacttac
      721 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      781 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      841 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      901 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      961 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     1021 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     1081 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     1141 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1201 tataagcaga gctaatcgtc gacgattcgt cagaatcggt cgccaccatg gtgagcaagg
     1261 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1321 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1381 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1441 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1501 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1561 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1621 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1681 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1741 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1801 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1861 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1921 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaattc tcgacctcga
     1981 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     2041 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     2101 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc
     2161 cgcggctcga gggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga
     2221 cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca
     2281 ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg
     2341 gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg
     2401 acgtgacaaa cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga
     2461 gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc
     2521 gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg
     2581 gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca
     2641 gtcggctccc tcgttgaccg aatcaccgac ctctctcccc agggggatcc accggagctt
     2701 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     2761 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     2821 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     2881 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     2941 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     3001 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     3061 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     3121 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     3181 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     3241 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     3301 tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg
     3361 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     3421 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     3481 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     3541 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     3601 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     3661 agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     3721 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3781 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3841 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     3901 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3961 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4021 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     4081 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     4141 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     4201 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     4261 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4321 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4381 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4441 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4501 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4561 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4621 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4681 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4741 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4801 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4861 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4921 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4981 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5041 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5101 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5161 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5221 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5281 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5341 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5401 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5461 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5521 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5581 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5641 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5701 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5761 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5821 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5881 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5941 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6001 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6061 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6121 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6181 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6241 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6301 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6361 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6421 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6481 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     6541 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     6601 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     6661 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     6721 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     6781 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     6841 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     6901 ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga
     6961 gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa
     7021 ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa
     7081 ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg
     7141 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     7201 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     7261 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     7321 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     7381 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     7441 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     7501 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
     7561 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     7621 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     7681 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     7741 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     7801 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
     7861 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     7921 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     7981 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     8041 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     8101 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     8161 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     8221 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     8281 ggagtaataa atctctggaa c
//
