LOCUS       sequence-385700-        7962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  control construct of TRE-VEGFA EF1a-rtTA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7962)
  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 7962)
  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: TRE-STOP EF1a-rtTA3
FEATURES             Location/Qualifiers
     source          1..7962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2028..2143
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     polyA_signal    2187..2321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(2510..2877)
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    2635..2653
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2671..2689
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2707..2725
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2743..2761
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2779..2797
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2815..2833
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2851..2869
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        2937..3148
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3170..3916
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    3930..4518
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3983..4003)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4401..4412)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4726..5359
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5487..5503)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5487..5503)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5500..5522)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5511..5527
                     /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(5535..5541,5542..5559,5560..5565))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5580..5601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5718..5735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5889..6477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5969..5988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6648..7439,7440..7508))
                     /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     7271..7290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7509..7613)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    7661..7795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2161 acagcagaga tccagtttat cgacttaact tgtttattgc agcttataat ggttacaaat
     2221 aaggcaatag catcacaaat ttcacaaata aggcattttt ttcactgcat tctagttttg
     2281 gtttgtccaa actcatcaat gtatcttatc atgtctggat ctcaaatccc tcggaagctg
     2341 cgcctgtctt aggttggagt gatacatttt tatcactttt acccgtcttt ggattaggca
     2401 gtagctctga cggccctcct gtcttaggtt agtgaaaaat gtcactctct tacccgtcat
     2461 tggctgtcca gcttagctcg caggggaggt ggtctggatc cttaaattct ttacgagggt
     2521 aggaagtggt acggaaagtt ggtataagac aaaagtgttg tggaattgaa gtttactcaa
     2581 aaaatcagca ctcttttata ggcgccctgg tttacataag caaagcttat acgttctcta
     2641 tcactgatag ggagtaaact ggatatacgt tctctatcac tgatagggag taaactgtag
     2701 atacgttctc tatcactgat agggagtaaa ctggtcatac gttctctatc actgataggg
     2761 agtaaactcc ttatacgttc tctatcactg atagggagta aagtctgcat acgttctcta
     2821 tcactgatag ggagtaaact cttcatacgt tctctatcac tgatagggag taaactcgag
     2881 gtgataattc cactaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
     2941 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg
     3001 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     3061 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     3121 tcgcaacggg tttgccgcca gaacacagga tcgatcacgt acggccacca tgtctagact
     3181 ggacaagagc aaagtcataa actctgctct ggaattactc aatggagtcg gtatcgaagg
     3241 cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca
     3301 cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc gagatgctgg acaggcatca
     3361 tacccactcc tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa
     3421 gtcataccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg
     3481 cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg
     3541 cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg
     3601 cgtattggag gaacaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga
     3661 ttctatgccc ccacttctga aacaagcaat tgagctgttc gaccggcagg gagccgaacc
     3721 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga
     3781 aagcggcggg ccgaccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc
     3841 ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga
     3901 catgctcccc gggtaaacgc gtctggaaca atcaacctct ggattacaaa atttgtgaaa
     3961 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4021 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4081 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4141 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4201 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     4261 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     4321 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     4381 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     4441 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     4501 tttgggccgc ctccccgcct ggaattaatt ctgcagtcga gacctagaaa aacatggagc
     4561 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga
     4621 ggaggaggag gtgggttttt ccagtcacac ctcaggtacc tttaagacca atgacttaca
     4681 aggcagctgt agatcttagc cactttttaa aagaaaagag gggactggaa gggctaattc
     4741 actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc
     4801 ctgattagca gaactacaca ccagggccag gggtcagata tccactgacc tttggatggt
     4861 gctacaagct agtaccagtt gagccagata aggtagaaga ggccaataaa ggagagaaca
     4921 ccagcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtgttag
     4981 agtggaggtt tgacagccgc ctagcatttc atcacgtggc ccgagagctg catccggagt
     5041 acttcaagaa ctgctgatat cgagcttgct acaagggact ttccgctggg gactttccag
     5101 ggaggcgtgg cctgggcggg actggggagt ggcgagccct cagatcctgc atataagcag
     5161 ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     5221 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     5281 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     5341 gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt
     5401 gcaaagaaat gaatatcaga gagtgagagg ccttgacatt gctagcgttt accgtcgacc
     5461 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5521 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5581 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5641 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5701 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5761 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5821 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5881 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5941 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6001 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6061 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6121 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6181 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6241 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6301 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6361 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6421 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6481 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6541 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6601 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6661 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6721 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6781 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6841 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6901 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6961 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7021 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7081 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7141 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7201 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7261 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7321 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7381 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7441 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7501 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7561 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7621 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc aacttgttta ttgcagctta
     7681 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     7741 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcaactg
     7801 gataactcaa gctaaccaaa atcatcccaa acttcccacc ccatacccta ttaccactgc
     7861 caattacctg tggtttcatt tactctaaac ctgtgattcc tctgaattat tttcatttta
     7921 aagaaattgt atttgttaaa tatgtactac aaacttagta gt
//
