LOCUS       sequence-8805-ex        9092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9092)
  AUTHORS   Pluta K, Luce MJ, Bao L, Agha-Mohammadi S, Reiser J
  TITLE     Tight control of transgene expression by lentivirus vectors 
            containing second-generation tetracycline-responsive promoters.
  JOURNAL   J Gene Med. 2005 Jun . 7(6):803-17.
  PUBMED    15655804
REFERENCE   2  (bases 1 to 9092)
  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: "J Gene Med.
            2005 Jun . 7(6):803-17."
COMMENT     Sequence Label: pNL-EGFP/TRETightdU3
FEATURES             Location/Qualifiers
     source          1..9092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             377..634
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      1260..1395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     misc_feature    1673..1906
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2091..2135
                     /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        2440..2754
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    2448..2466
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2484..2502
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2519..2537
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2555..2573
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2591..2609
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2626..2644
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2662..2680
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2727..2751
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(2769..2771,2772..2774,2775..3485)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2769..2771,2772..2774,2775..3485)
                     /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(2814..2835)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3075..3094)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3422..3443
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3690..3922
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5564..5581)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5735..6323)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5815..5834)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6494..7285,7286..7354))
                     /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     7117..7136
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7355..7459)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7527..7545
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7596..7618)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    7704..7842
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     7757..7776
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tggaagggct aatttggtcc caaaaaagac aagagatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac
      121 tgacctttgg atggtgcttc aagttagtac cagttgaacc agagcaagta gaagaggcca
      181 aataaggaga gaagaacagc ttgttacacc ctatgagcca gcatgggatg gaggacccgg
      241 agggagaagt attagtgtgg aagtttgaca gcctcctagc atttcgtcac atggcccgag
      301 agctgcatcc ggagtactac aaagactgct gacatcgagc tttctacaag ggactttccg
      361 ctggggactt tccagggagg tgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 gctacatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgctca aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagtaaa gccagaggag atctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcggta ttaagcgggg gagaattaga taaatgggaa
      841 aaaattcggt taaggccagg gggaaagaaa caatataaac taaaacatat agtatgggca
      901 agcagggagc tagaacgatt cgcagttaat cctggccttt tagagacatc agaaggctgt
      961 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
     1021 ttatataata caatagcagt cctctattgt gtgcatcaaa ggatagatgt aaaagacacc
     1081 aaggaagcct tagataagat agaggaagag caaaacaaag taagaaaaag gcacagcaag
     1141 cagcagctga cacaggaaac aacagccagg tcagccaaaa ttaccctata gtgcagaacc
     1201 tccaggggca aatggtacat caggccatat cacctagaac tttaaatatg actccgccca
     1261 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     1321 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     1381 gcttttttgg aggcctaggc ttttgcaaat gcataaaaga aaagggggga ttggggggta
     1441 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     1501 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccaca
     1561 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1621 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1681 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1741 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1801 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1861 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1921 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1981 ctctggaaca gatttggaat aacatgacct ggatggagtg ggacagagaa attaacaatt
     2041 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     2101 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     2161 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     2221 tttttgctgt actttctgta gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     2281 agacccacct cccaatcccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     2341 gagagagaga cagagacaga tccattcgat tagtgaacgg atctagttcc tcccaccgta
     2401 cacgcctacc tcgacccatc ggccctttcg tcttcactcg agtttactcc ctatcagtga
     2461 tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgatgtc gagtttactc
     2521 cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg
     2581 tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttatccct atcagtgata
     2641 gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg aggtaggcgt
     2701 gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat cgcctgggat
     2761 ccccgcggat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2821 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2881 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2941 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     3001 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     3061 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     3121 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     3181 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     3241 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     3301 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     3361 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     3421 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     3481 acaagtccgg actcagatct ctcgagacct agaaaaacat ggagcaatca caagtagcaa
     3541 tacagcagct aacaatgctg cttgtgcctg gctagaagca caagaggagg aagaggtggg
     3601 ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct
     3661 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aaagaagaca
     3721 aagctgcttt ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     3781 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     3841 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     3901 tcagtgtgga aaatctctag caccccccag gaggtagagg ttgcagtgag ccaagatcgc
     3961 gccactgcat tccagcctgg gcaagaaaac aagactgtct aaaataataa taataagtta
     4021 agggtattaa atatatttat acatggaggt cataaaaata tatatatttg ggctgggcgc
     4081 agtggctcac acctgcgccc ggccctttgg gaggccgagg caggtggatc acctgagttt
     4141 gggagttcca gaccagcctg accaacatgg agaaacccct tctctgtgta tttttagtag
     4201 attttatttt atgtgtattt tattcacagg tatttctgga aaactgaaac tgtttttcct
     4261 ctactctgat accacaagaa tcatcagcac agaggaagac ttctgtgatc aaatgtggtg
     4321 ggagagggag gttttcacca gcacatgagc agtcagttct gccgcagact cggcgggtgt
     4381 ccttcggttc agttccaaca ccgcctgcct ggagagaggt cagaccacag ggtgagggct
     4441 cagtccccaa gacataaaca cccaagacat aaacacccaa caggtccacc ccgcctgctg
     4501 cccaggcaga gccgattcac caagacggga attaggatag agaaagagta agtcacacag
     4561 agccggctgt gcgggagaac ggagttctat tatgactcaa atcagtctcc ccaagcattc
     4621 ggggatcaga gtttttaagg ataacttagt gtgtaggggg ccagtgagtt ggagatgaaa
     4681 gcgtagggag tcgaaggtgt ccttttgcgc cgagtcagtt cctgggtggg ggccacaaga
     4741 tcggatgagc cagtttatca atccgggggt gccagctgat ccatggagtg cagggtctgc
     4801 aaaatatctc aagcactgat tgatcttagg ttttacaata gtgatgttac cccaggaaca
     4861 atttggggaa ggtcagaatc ttgtagcctg tagctgcatg actcctaaac cataatttct
     4921 tttttgtttt ttttttttta tttttgagac agggtctcac tctgtcacct aggctggagt
     4981 gcagtggtgc aatcacagct cactgcagcc tcaacgtcgt aagctcaagc gatcctccca
     5041 cctcagcctg cctggtagct gagactacaa gcgacgcccc agttaatttt tgtatttttg
     5101 gtagaggcag cgttttgccg tgtggccctg gctggtctcg aactcctggg ctcaagtgat
     5161 ccagcctcag cctcccaaag tgctgggaca accggggcca gtcactgcac ctggccctaa
     5221 accataattt ctaatctttt ggctaatttg ttagtcctac aaaggcagtc tagtccccag
     5281 gcaaaaaggg ggtttgtttc gggaaagggc tgttactgtc tttgtttcaa actataaact
     5341 aagttcctcc taaacttagt tcggcctaca cccaggaatg aacaaggaga gcttggaggt
     5401 tagaagcacg atggaattgg ttaggtcaga tctctttcac tgtctgagtt ataattttgc
     5461 aatggtggtt caaagactgc ccgcttctga caccagtcgc tgcattaatg aatcggccaa
     5521 cgcgcgggga gaggcggttt gcgtattggc gctcttccgc ttcctcgctc actgactcgc
     5581 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5641 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5701 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5761 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5821 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5881 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct
     5941 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6001 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6061 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6121 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     6181 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6241 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6301 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6361 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6421 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6481 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6541 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6601 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6661 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6721 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6781 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6841 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6901 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6961 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7021 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7081 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7141 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7201 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7261 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7321 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7381 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7441 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     7501 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag
     7561 aactgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     7621 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     7681 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     7741 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     7801 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     7861 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccaggggagg
     7921 cagagattgc agtaagctga gatcgcagca ctgcactcca gcctgggcga cagagtaaga
     7981 ctctgtctca aaaataaaat aaataaatca atcagatatt ccaatctttt cctttattta
     8041 tttatttatt ttctattttg gaaacacagt ccttccttat tccagaatta cacatatatt
     8101 ctatttttct ttatatgctc cagttttttt tagaccttca cctgaaatgt gtgtatacaa
     8161 aatctaggcc agtccagcag agcctaaagg taaaaaataa aataataaaa aataaataaa
     8221 atctagctca ctccttcaca tcaaaatgga gatacagctg ttagcattaa ataccaaata
     8281 acccatcttg tcctcaataa ttttaagcgc ctctctccac cacatctaac tcctgtcaaa
     8341 ggcatgtgcc ccttccgggc gctctgctgt gctgccaacc aactggcatg tggactctgc
     8401 agggtcccta actgccaagc cccacagtgt gccctgaggc tgccccttcc ttctagcggc
     8461 tgcccccact cggctttgct ttccctagtt tcagttactt gcgttcagcc aaggtctgaa
     8521 actaggtgcg cacagagcgg taagactgcg agagaaagag accagcttta cagggggttt
     8581 atcacagtgc accctgacag tcgtcagcct cacagggggt ttatcacatt gcaccctgac
     8641 agtcgtcagc ctcacagggg gtttatcaca gtgcaccctt acaatcattc catttgattc
     8701 acaatttttt tagtctctac tgtgcctaac ttgtaagtta aatttgatca gaggtgtgtt
     8761 cccagagggg aaaacagtat atacagggtt cagtactatc gcatttcagg cctccacctg
     8821 ggtcttggaa tgtgtccccc gaggggtgat gactacctca gttggatctc cacaggtcac
     8881 agtgacacaa gataaccaag acacctccca aggctaccac aatgggccgc cctccacgtg
     8941 cacatggccg gaggaactgc catgtcggag gtgcaagcac acctgcgcat cagagtcctt
     9001 ggtgtggagg gagggaccag cgcagcttcc agccatccac ctgatgaaca gaacctaggg
     9061 aaagccccag ttctacttac accaggaaag gc
//
