LOCUS       sequence-474986-        7484 bp    DNA     circular SYN 06-JUN-2025
DEFINITION  doxycycline inducible multicistronic lentiviral gene expression 
            system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7484)
  TITLE     pUltra plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7484)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pUltra-dox
FEATURES             Location/Qualifiers
     source          1..7484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1058..1284
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1285..1465
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1512..1637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2130..2363
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2548..2592
                     /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    2890..3007
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    3054..3087
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3102..3120
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3144..3162
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3186..3204
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3228..3246
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3270..3288
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        3321..3359
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3356..3380
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3466..3475
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3472..4173
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(3605..3623)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3873..3892
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4067..4090
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             4183..4239
                     /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             4261..4314
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    4336..4369
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    4381..4969
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4434..4454)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4852..4863)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5060..5293
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5365..5486
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5402..5421)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5456..5475
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5526..5661
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5681..5700)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5682..5700)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5710..5727)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5710..5726)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5719..5741)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5868..6323
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5955..5974)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6165..6186
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6349..6453
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6454..6522,6523..7314)
                     /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     complement(6672..6691)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctcact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
     1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
     1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
     1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2761 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     2821 attcgattag tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt
     2881 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2941 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     3001 aaattttcgg gtttattaca gggacagcag agatccagtt tggttaatta acgataactt
     3061 cgtataatgt gtactatacg aagttatctc gagtttacca ctccctatca gtgatagaga
     3121 aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagttt
     3181 accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga
     3241 tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc
     3301 gagctcggta cccgggtcga ggtaggcgtg tacggtggga ggcctatata agcagagctc
     3361 gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa
     3421 gacaccggga ccgatccagc ctccgcggac cggtggatcg atcccgccac catggtgagc
     3481 aagggcgagg aggtcatcaa agagttcatg cgcttcaagg tgcgcatgga gggctccatg
     3541 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     3601 accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc
     3661 ccccagttca tgtacggctc caaggcgtac gtgaagcacc ccgccgacat ccccgattac
     3721 aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     3781 ggtctggtga ccgtgaccca ggactcctcc ctgcaggacg gcacgctgat ctacaaggtg
     3841 aagatgcgcg gcaccaactt cccccccgac ggccccgtaa tgcagaagaa gaccatgggc
     3901 tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagatccac
     3961 caggccctga agctgaagga cggcggccac tacctggtgg agttcaagac catctacatg
     4021 gccaagaagc ccgtgcaact gcccggctac tactacgtgg acaccaagct ggacatcacc
     4081 tcccacaacg aggactacac catcgtggaa cagtacgagc gctccgaggg ccgccaccac
     4141 ctgttcctgt acggcatgga cgagctgtac aagggatctg gagcaacaaa cttctcacta
     4201 ctcaaacaag caggtgacgt ggaggagaat cccgggcctt ctagaggatc cggctccggc
     4261 gagggcaggg gaagtctact aacatgcggg gacgtggagg aaaatcccgg cccagctagc
     4321 gtgtgatcag aattcataac ttcgtataat gtgtactata cgaagttatg tcgactcgac
     4381 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     4441 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     4501 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     4561 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     4621 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     4681 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     4741 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
     4801 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     4861 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcatctt
     4921 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggaattaat
     4981 tcgagctcgg tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     5041 ttaaaagaaa aggggggact ggaagggcta cgtaactccc aacgaagaca agatctgctt
     5101 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     5161 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     5221 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     5281 aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     5341 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     5401 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5461 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     5521 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5581 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     5641 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     5701 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5761 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5821 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     5881 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5941 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     6001 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     6061 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     6121 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     6181 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     6241 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     6301 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     6361 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6421 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6481 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6541 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6601 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6661 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6721 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6781 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6841 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6901 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6961 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     7021 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     7081 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     7141 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     7201 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     7261 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     7321 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7381 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7441 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
