LOCUS       sequence-385687-        7149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct of hPGK driven rtTA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7149)
  AUTHORS   Yuan S, Stewart KS, Yang Y, Abdusselamoglu MD, Parigi SM, Feinberg 
            TY, Tumaneng K, Yang H, Levorse JM, Polak L, Ng D, Fuchs E
  TITLE     Ras drives malignancy through stem cell crosstalk with the 
            microenvironment.
  JOURNAL   Nature. 2022 Nov 30. doi: 10.1038/s41586-022-05475-6.
  PUBMED    36450983
REFERENCE   2  (bases 1 to 7149)
  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: "Nature. 
            2022 Nov 30. doi: 10.1038/s41586-022-05475-6."
COMMENT     Sequence Label: hPGK-rtTA3
FEATURES             Location/Qualifiers
     source          1..7149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     426..444
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             529..1236
                     /codon_start=1
                     /product="reverse tetracycline transactivator 3"
                     /label=rtTA3
                     /note="activates expression from Tet-responsive promoters 
                     in the presence of doxycycline"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPG"
     LTR             1322..1555
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1627..1748
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1664..1683)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1718..1737
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1788..1923
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1943..1962)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1944..1962)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1972..1989)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1972..1988)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1981..2003)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2130..2585
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2217..2236)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2427..2448
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2611..2715
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2716..2784,2785..3576)
                     /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(2934..2953)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3747..4335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4236..4255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4489..4506
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4623..4644
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4659..4664,4665..4682,4683..4689)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4697..4713
                     /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     4702..4724
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4721..4737
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4721..4737
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4756..4776
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4758..4776
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4804..5030
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5031..5211
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5258..5383
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5876..6109
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6294..6338
                     /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        6636..6884
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6636..6656
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6807..6826
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    6980..7097
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7146..7149,1..507)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
       61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
      121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
      181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
      241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
      301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
      361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
      421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
      481 gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat gtctaggctg
      541 gacaagagca aagtcataaa cggagctctg gaattactca atggtgtcgg tatcgaaggc
      601 ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc agcctaccct gtactggcac
      661 gtgaagaaca agcgggccct gctcgatgcc ctgccaatcg agatgctgga caggcatcat
      721 acccacttct gccccctgga aggcgagtca tggcaagact ttctgcggaa caacgccaag
      781 tcataccgct gtgctctcct ctcacatcgc gacggggcta aagtgcatct cggcacccgc
      841 ccaacagaga aacagtacga aaccctggaa aatcagctcg cgttcctgtg tcagcaaggc
      901 ttctccctgg agaacgcact gtacgctctg tccgccgtgg gccactttac actgggctgc
      961 gtattggagg aacaggagca tcaagtagca aaagaggaaa gagagacacc taccaccgat
     1021 tctatgcccc cacttctgag acaagcaatt gagctgttcg accggcaggg agccgaacct
     1081 gccttccttt tcggcctgga actaatcata tgtggcctgg agaaacagct aaagtgcgaa
     1141 agcggcgggc cgaccgacgc ccttgacgat tttgacttag acatgctccc agccgatgcc
     1201 cttgacgatt ttgaccttga catgctcccc gggtaaatgc atgtttaaac gactgcttta
     1261 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     1321 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     1381 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     1441 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     1501 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt
     1561 agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt
     1621 gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1681 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     1741 gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa
     1801 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     1861 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     1921 gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
     1981 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     2041 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     2101 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     2161 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2221 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2281 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2341 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2401 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2461 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2521 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     2581 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2641 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2701 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2761 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2821 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2881 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2941 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3001 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3061 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3121 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3181 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3241 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3301 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3361 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3421 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3481 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3541 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3601 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3661 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3721 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3781 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3841 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     3901 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3961 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4021 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4081 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4141 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4201 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4261 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4321 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4381 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4441 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4501 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     4561 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4621 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     4681 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     4741 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct
     4801 taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac
     4861 atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg
     4921 atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga accactgaat
     4981 tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct
     5041 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     5101 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     5161 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
     5221 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
     5281 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
     5341 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
     5401 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
     5461 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     5521 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     5581 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     5641 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     5701 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     5761 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     5821 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     5881 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     5941 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     6001 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     6061 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     6121 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     6181 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     6241 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     6301 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     6361 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     6421 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     6481 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     6541 gtggagagag agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat
     6601 cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt
     6661 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     6721 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     6781 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     6841 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggcaac aagatgaaga
     6901 gcaccaactc gagttggtgc tcttcatctt gttgttttta attctcgacc tcgagacaaa
     6961 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     7021 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     7081 caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt tggccgcggc
     7141 tcgaggggg
//
