LOCUS       sequence-275844-        7360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable and temporal expression control of SPI1 and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7360)
  TITLE     Xlone Puro plasmids for inducible gene expression in mammal cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7360)
  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: 
            "Unpublished"
COMMENT     Sequence Label: XLone-Puro SPI1 P2A mCherry
FEATURES             Location/Qualifiers
     source          1..7360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        34..245
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      265..274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             274..297
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             298..1041
                     /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"
     CDS             1051..1104
                     /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"
     CDS             1114..1713
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1114..1133)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1610..1630
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1734..1815
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1991..2053
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2079..2095)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2079..2095)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2092..2114)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2103..2119
                     /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(2127..2133,2134..2151,2152..2157))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2172..2193
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2310..2327)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2481..3069)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2561..2580)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3240..4031,4032..4100))
                     /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     3863..3882
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4101..4205)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4273..4291
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4329..4351)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4451..4470
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4664..4686
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4678..4695
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4679..4695
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   4706..4740
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5016..5150
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5356..6063)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(5634..5653)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5903..5921
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(6064..6087)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(6091..6147)
                     /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"
     promoter        complement(6985..7352)
                     /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    7110..7128
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7146..7164
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7182..7200
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7218..7236
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7254..7272
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7290..7308
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7326..7344
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aattccactc gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
       61 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
      121 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
      181 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
      241 cacaggtgtc gtgacgcggg atccgccacc atggattaca aagacgatga cgataagatg
      301 tctagactgg acaagagcaa agtcataaac tctgctctgg aattactcaa tggagtcggt
      361 atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg
      421 tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga gatgctggac
      481 aggcatcata cccactcctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac
      541 aacgccaagt cataccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc
      601 ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt
      661 cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca
      721 ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag agagacacct
      781 accaccgatt ctatgccccc acttctgaaa caagcaattg agctgttcga ccggcaggga
      841 gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta
      901 aagtgcgaaa gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga catgctccca
      961 gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt
     1021 gaccttgaca tgctccccgg gggcagcgga gagggcagag gaagtcttct aacatgcggt
     1081 gacgtggagg agaatcccgg ccctaggctc gagatgaccg agtacaagcc cacggtgcgc
     1141 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     1201 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     1261 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     1321 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     1381 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     1441 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     1501 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     1561 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     1621 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     1681 acctggtgca tgacccgcaa gcccggtgcc tgatctagag ggcccgttta gttaacttgt
     1741 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     1801 catttttttc actgctaaaa gttttgttac tttatagaag aaattttgag tttttgtttt
     1861 tttttaataa ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag
     1921 tgtaaatata ataaaactta atatctattc aaattaataa ataaacctcg atatacagac
     1981 cgataaaaca catgcgtcaa ttttacgcat gattatcttt aacgtacgtc acaatatgat
     2041 tatctttcta gggttaagtc gaccttggcg taatcatggt catagctgtt tcctgtgtga
     2101 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2161 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2221 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2281 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2341 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2401 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2461 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2521 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2581 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2641 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2701 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2761 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2821 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2881 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     2941 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3001 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3061 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3121 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3181 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3241 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3301 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3361 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3421 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3481 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3541 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3601 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3661 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3721 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3781 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3841 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3901 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3961 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4021 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4081 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4141 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4201 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     4261 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     4321 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     4381 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     4441 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     4501 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
     4561 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     4621 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     4681 aaaacgacgg ccagtgaatt cttaacccta gaaagatagt ctgcgtaaaa ttgacgcatg
     4741 cattcttgaa atattgctct ctctttctaa atagcgcgaa tccgtcgctg tgcatttagg
     4801 acatctcagt cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg taagtgtcac
     4861 tgattttgaa ctataacgac cgcgtgagtc aaaatgacgc atgattatct tttacgtgac
     4921 ttttaagatt taactcatac gataattata ttgttatttc atgttctact tacgtgataa
     4981 cttattatat atatattttc ttgttataga tatcaaactt gtttattgca gcttataatg
     5041 gttacaaata aggcaatagc atcacaaatt tcacaaataa ggcatttttt tcactgcatt
     5101 ctagttttgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tcaaatccct
     5161 cggaagctgc gcctgtctta ggttggagtg atacattttt atcactttta cccgtctttg
     5221 gattaggcag tagctctgac ggccctcctg tcttaggtta gtgaaaaatg tcactctctt
     5281 acccgtcatt ggctgtccag cttagctcgc aggggaggtg gtctactagt gggttattga
     5341 atatgatcgg aattcttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc
     5401 ggcgcgttcg tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt
     5461 gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt
     5521 gacctcagcg tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt
     5581 cagggcgccg tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt
     5641 ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa
     5701 ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt
     5761 catcacgcgc tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc
     5821 ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca
     5881 ggcgaagggc agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta
     5941 ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat
     6001 gtgcaccttg aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct
     6061 caccttgtca tcgtcgtcct tgtagtccat tggcccggga ttctcttcga catcccctgc
     6121 ttgtttcaac agggagaagt tagtggcgcc gctgccgcta gcgtggggcg ggtggcgccg
     6181 ctcggccagg cccccgcggc ccagcacttc gccgctgaac tggtaggtga gcttcttctt
     6241 caccttcttg acctcgcccg tcttgccgta gttgcgcagc gcgcgcgcca tcttctggta
     6301 ggtcatcttc ttgcggttgc ccttctggat gccccagcgg tgcgccagcg cctccttgtg
     6361 cttggacgag aactggaagg tgcccttgtc cttgtccacc caccagatgc tgtccttcat
     6421 gtcgccgctg cggagcaggt ccaacaggaa ctggtacagg cggatcttct tcttgctgcc
     6481 tgtctcccca ggcaggagcc caggcccggg ctccaggcca tccgcctcgc cgtcagacac
     6541 ctccagtggg gggctctgcc gctcgccctc ctcctcatct gagctgggct gggctgggga
     6601 cagggatggg tactggaggc acatccgggg caggtaggag acctggtggc caagactggg
     6661 atggggtggc accatggggg tatcgaggac gtgcatctgc tccagctcca tgtggcggta
     6721 gagctgctgc agctgcgggg gctgcacgct ctggagctcc gtgaagttgt tctcggcgaa
     6781 gctctcgaac tcgctgtgca cgtggtgggg gtggaagtcc cagtaatggt cgctatggct
     6841 ctccccatca ctgctgagat aggggtaata ctcgtgcgtt tggcgttggt atagatccgt
     6901 gtcatagggc accaggtctt ctgatggagg ggggacgagg ggaaaccctt ccattttgca
     6961 cgcctgtaac atggtggcgg tacctttacg agggtaggaa gtggtacgga aagttggtat
     7021 aagacaaaag tgttgtggaa ttgaagttta ctcaaaaaat cagcactctt ttataggcgc
     7081 cctggtttac ataagcaaag cttatacgtt ctctatcact gatagggagt aaactggata
     7141 tacgttctct atcactgata gggagtaaac tgtagatacg ttctctatca ctgataggga
     7201 gtaaactggt catacgttct ctatcactga tagggagtaa actccttata cgttctctat
     7261 cactgatagg gagtaaagtc tgcatacgtt ctctatcact gatagggagt aaactcttca
     7321 tacgttctct atcactgata gggagtaaac tcgaggtgat
//
