pTre3G-mScarletI-iPAK4-CMV-rtTA3

LOCUS       pTre3G-mScarletI-iPAK4-CMV-rtTA3               6104 bp DNA     circular SYN 23-APR-2024
DEFINITION  Vector for doxycycline-inducible expression of two genes in cells 
            containing the Tet-On(R) 3G transactivator protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6104)
  AUTHORS   Clontech (TaKaRa)
  TITLE     Direct Submission
  JOURNAL   Exported Jul 13, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..6104
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          1201..2277
                     /mol_type="genomic DNA"
                     /organism="."
     promoter        1..451
                     /label=TRE3G BI promoter
                     /note="3rd-generation Tet-responsive bidirectional promoter
                     that can be activated by binding of Tet-On 3G"
     primer_bind     complement(58..75)
                     /label=Ptre3G-R
     protein_bind    81..99
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    117..135
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    153..171
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    189..207
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    225..243
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    261..279
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    297..315
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     430..447
                     /label=Ptre3G-F
     primer_bind     458..510
                     /label=BamHI-iPAK4-F
     primer_bind     477..519
                     /label=AgeI-EGFP-F
     primer_bind     479..524
                     /label=mScarletI-1P0K-F
     primer_bind     complement(495..521)
                     /label=mScarletI-FLAG-Qch-R
     regulatory      499..508
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             505..1200
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(638..656)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     906..925
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1179..1226
                     /label=GFP-iPAK4-F
     primer_bind     1179..1226
                     /label=Primer 1a
     primer_bind     complement(1180..1226)
                     /label=XFP-iPAK4-R
     primer_bind     complement(1180..1226)
                     /label=XFP-iPAK4-Ra
     CDS             1213..1326
                     /codon_start=1
                     /label=INKA-box
                     /translation="EAEDWTAALLNRGRSRQPLVLGDNCFADLVHNWMELPE"
     CDS             1333..2274
                     /codon_start=1
                     /label=Pak4cat
                     /translation="PAAPAVPGPPGPRSPQREPQRVSHEQFRAALQLVVDPGDPRSYLD
                     NFIKIGEGSTGIVCIATVRSSGKLVAVKKMDLRKQQRRELLFNEVVIMRDYQHENVVEM
                     YNSYLVGDELWVVMEFLEGGALTDIVTHTRMNEEQIAAVCLAVLQALSVLHAQGVIHRD
                     IKSDSILLTHDGRVKLSDFGFCAQVSKEVPRRKSLVGTPYWMAPELISRLPYGPEVDIW
                     SLGIMVIEMVDGEPPYFNEPPLKAMKMIRDNLPPRLKNLHKVSPSLKGFLDRLLVRDPA
                     QRATAAELLKHPFLAKAGPPASIVPLMRQNRTR"
     primer_bind     complement(1426..1444)
                     /label=PAK4-R
     primer_bind     complement(2257..2301)
                     /label=NotI-Pak4-R
     polyA_signal    2410..2491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2410..2428
                     /label=sv40-polyA(YK)
     primer_bind     complement(2551..2574)
                     /label=PTRE-R（A3254）(YK)
     rep_origin      complement(2673..3261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3432..4292)
                     /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     4206..4226
                     /label=Amp-R-Seq
     promoter        complement(4293..4397)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4430..4511
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4493..4511)
                     /label=sv40-polyA(YK)
     primer_bind     4625..4672
                     /label=TetOn3G-NheI-R
     CDS             complement(4649..5395)
                     /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"
     promoter        complement(5498..5701)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        5702..6081
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(6063..6101)
                     /label=CMV-KpnI-F
ORIGIN
        1 gcgatctgac ggttcactaa acgagctctg cttatatagg cctcccaccg tacacgccac
       61 ctcgacatac tcgagtttac tccctatcag tgatagagaa cgtatgaaga gtttactccc
      121 tatcagtgat agagaacgta tgcagacttt actccctatc agtgatagag aacgtataag
      181 gagtttactc cctatcagtg atagagaacg tatgaccagt ttactcccta tcagtgatag
      241 agaacgtatc tacagtttac tccctatcag tgatagagaa cgtatatcca gtttactccc
      301 tatcagtgat agagaacgta taagctttag gcgtgtacgg tgggcgccta taaaagcaga
      361 gctcgtttag tgaaccgtca gatcgcctgg agcaattcca caacactttt gtcttatacc
      421 aactttccgt accacttcct accctcgtaa agtcgacacc ggggcccaga tctccgcggg
      481 gatccatcga taccggtcgc caccatggtg agcaagggcg aggcagtgat caaggagttc
      541 atgcggttca aggtgcacat ggagggctcc atgaacggcc acgagttcga gatcgagggc
      601 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
      661 ggccccctgc ccttctcctg ggacatcctg tcccctcagt tcatgtacgg ctccagggcc
      721 ttcatcaagc accccgccga catccccgac tactataagc agtccttccc cgagggcttc
      781 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc
      841 tccctggagg acggcaccct gatctacaag gtgaagctcc gcggcaccaa cttccctcct
      901 gacggccccg taatgcagaa gaagacaatg ggctgggaag cgtccaccga gcggttgtac
      961 cccgaggacg gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa ggacggcggc
     1021 cgctacctgg cggacttcaa gaccacctac aaggccaaga agcccgtgca gatgcccggc
     1081 gcctacaacg tcgaccgcaa gttggacatc acctcccaca acgaggacta caccgtggtg
     1141 gaacagtacg aacgctccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     1201 tcaggtggat ccgaagcaga ggactggaca gcagccctac tgaacagggg tcgcagtcgc
     1261 cagcccctgg tactagggga caattgcttt gctgacttgg tgcacaactg gatggagctg
     1321 cctgaggaat tccccgccgc ccctgctgtt cctgggcccc ctggcccccg ctcaccacag
     1381 cgggagccac agcgagtatc ccatgagcag ttccgggctg ccctgcagct ggtggtggac
     1441 ccaggcgacc cccgctccta cctggacaac ttcatcaaga ttggcgaggg ctccacgggc
     1501 atcgtgtgca tcgccaccgt gcgcagctcg ggcaagctgg tggccgtcaa gaagatggac
     1561 ctgcgcaagc agcagaggcg cgagctgctc ttcaacgagg tggtaatcat gagggactac
     1621 cagcacgaga atgtggtgga gatgtacaac agctacctgg tgggggacga gctctgggtg
     1681 gtcatggagt tcctggaagg aggcgccctc accgacatcg tcacccacac caggatgaac
     1741 gaggagcaga tcgcagccgt gtgccttgca gtgctgcagg ccctgtcggt gctccacgcc
     1801 cagggcgtca tccaccggga catcaagagc gactcgatcc tgctgaccca tgatggcagg
     1861 gtgaagctgt cagactttgg gttctgcgcc caggtgagca aggaagtgcc ccgaaggaag
     1921 tcgctggtcg gcacgcccta ctggatggcc ccagagctca tctcccgcct tccctacggg
     1981 ccagaggtag acatctggtc gctggggata atggtgattg agatggtgga cggagagccc
     2041 ccctacttca acgagccacc cctcaaagcc atgaagatga ttcgggacaa cctgccaccc
     2101 cgactgaaga acctgcacaa ggtgtcgcca tccctgaagg gcttcctgga ccgcctgctg
     2161 gtgcgagacc ctgcccagcg ggccacggca gccgagctgc tgaagcaccc attcctggcc
     2221 aaggcagggc cgcctgccag catcgtgccc ctcatgcgcc agaaccgcac cagatgacgc
     2281 ggccgccggc gatatctcca gaggatcata atcagccata ccacatttgt agaggtttta
     2341 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     2401 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2461 aatttcacaa ataaagcatt tttttcactg ccccgagctt cctcgctcac tgactcgctg
     2521 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2581 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2641 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2701 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2761 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2821 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt
     2881 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2941 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3001 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3061 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     3121 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3181 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3241 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3301 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3361 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3421 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3481 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3541 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3601 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3661 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3721 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3781 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3841 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3901 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3961 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4021 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4081 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4141 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4201 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4261 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4321 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4381 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcgg cagtgaaaaa
     4441 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     4501 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     4561 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc
     4621 tagacatatg ctgcagacgc gtgctagctt acccggggag catgtcaagg tcaaaatcgt
     4681 caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca
     4741 tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt
     4801 tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc
     4861 ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct
     4921 ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc agtgtaaagt
     4981 ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga
     5041 acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca
     5101 ctttagcccc gtcgcgatgt gagaggagag cacagcggta tgacttggcg ttgttccgca
     5161 gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca
     5221 tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag
     5281 gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactc
     5341 cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta gacatggtga
     5401 attcggggcc gcggaggctg gatcggtccc ggtgtcttct atggaggtca aaacagcgtg
     5461 gatggcgtct ccaggcgatc tgacggttca ctaaacgagc tctgcttata tagacctccc
     5521 accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa
     5581 agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg
     5641 gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca
     5701 ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg
     5761 tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg
     5821 tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca
     5881 cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg
     5941 acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg
     6001 taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat
     6061 aactagtcaa taatcaatgt cggtaccccc ggggaattct ccag
//