LOCUS       sequence-275874-        7249 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 7249)
  TITLE     Xlone Puro plasmids for inducible gene expression in mammal cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7249)
  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            "Unpublished"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-BSD SPI1 P2A mCherry
FEATURES             Location/Qualifiers
     source          1..7249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /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    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1214..1270
                     /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             1274..1297
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1298..2005
                     /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(1440..1458)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1708..1727
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2211..2345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2621..2655
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2666..2683)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2666..2682)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2675..2697)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2891..2910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3010..3032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3070..3088)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3156..3260
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3261..3329,3330..4121)
                     /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(3479..3498)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4292..4880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4781..4800
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5034..5051
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5168..5189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5204..5209,5210..5227,5228..5234)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5242..5258
                     /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     5247..5269
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5266..5282
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5266..5282
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5308..5370
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5546..5627
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5744..6142)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(6143..6199)
                     /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             complement(6209..6952)
                     /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             complement(6953..6976)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6976..6985
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7005..7216)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta ccgccaccat gttacaggcg tgcaaaatgg aagggtttcc
      421 cctcgtcccc cctccatcag aagacctggt gccctatgac acggatctat accaacgcca
      481 aacgcacgag tattacccct atctcagcag tgatggggag agccatagcg accattactg
      541 ggacttccac ccccaccacg tgcacagcga gttcgagagc ttcgccgaga acaacttcac
      601 ggagctccag agcgtgcagc ccccgcagct gcagcagctc taccgccaca tggagctgga
      661 gcagatgcac gtcctcgata cccccatggt gccaccccat cccagtcttg gccaccaggt
      721 ctcctacctg ccccggatgt gcctccagta cccatccctg tccccagccc agcccagctc
      781 agatgaggag gagggcgagc ggcagagccc cccactggag gtgtctgacg gcgaggcgga
      841 tggcctggag cccgggcctg ggctcctgcc tggggagaca ggcagcaaga agaagatccg
      901 cctgtaccag ttcctgttgg acctgctccg cagcggcgac atgaaggaca gcatctggtg
      961 ggtggacaag gacaagggca ccttccagtt ctcgtccaag cacaaggagg cgctggcgca
     1021 ccgctggggc atccagaagg gcaaccgcaa gaagatgacc taccagaaga tggcgcgcgc
     1081 gctgcgcaac tacggcaaga cgggcgaggt caagaaggtg aagaagaagc tcacctacca
     1141 gttcagcggc gaagtgctgg gccgcggggg cctggccgag cggcgccacc cgccccacgc
     1201 tagcggcagc ggcgccacta acttctccct gttgaaacaa gcaggggatg tcgaagagaa
     1261 tcccgggcca atggactaca aggacgacga tgacaaggtg agcaagggcg aggaggataa
     1321 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
     1381 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     1441 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     1501 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     1561 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     1621 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     1681 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     1741 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
     1801 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     1861 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
     1921 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
     1981 cggcatggac gagctgtaca agtaagaatt ccgatcatat tcaataaccc actagtagac
     2041 cacctcccct gcgagctaag ctggacagcc aatgacgggt aagagagtga catttttcac
     2101 taacctaaga caggagggcc gtcagagcta ctgcctaatc caaagacggg taaaagtgat
     2161 aaaaatgtat cactccaacc taagacaggc gcagcttccg agggatttga gatccagaca
     2221 tgataagata cattgatgag tttggacaaa ccaaaactag aatgcagtga aaaaaatgcc
     2281 ttatttgtga aatttgtgat gctattgcct tatttgtaac cattataagc tgcaataaac
     2341 aagtttgata tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat
     2401 gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat
     2461 gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac
     2521 aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga
     2581 ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag
     2641 actatctttc tagggttaag aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     2701 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     2761 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     2821 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     2881 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     2941 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3001 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3061 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3121 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3181 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3241 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3301 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3361 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3421 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3481 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     3541 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3601 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3661 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3721 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3781 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     3841 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3901 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3961 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4021 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4081 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4141 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4201 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4261 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4321 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4381 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4441 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4501 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4561 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4621 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4681 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4741 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4801 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4861 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4921 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4981 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5041 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     5101 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     5161 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     5221 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     5281 accatgatta cgccaaggtc gacttaaccc tagaaagata atcatattgt gacgtacgtt
     5341 aaagataatc atgcgtaaaa ttgacgcatg tgttttatcg gtctgtatat cgaggtttat
     5401 ttattaattt gaatagatat taagttttat tatatttaca cttacatact aataataaat
     5461 tcaacaaaca atttatttat gtttatttat ttattaaaaa aaaacaaaaa ctcaaaattt
     5521 cttctataaa gtaacaaaac ttttagcagt gaaaaaaatg ctttatttgt gaaatttgtg
     5581 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     5641 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     5701 acctctacaa atgtggtatg gctgattatg atcctctgga gatttagccc tcccacacat
     5761 aaccagaggg cagcaattca cgaatcccaa ctgccgtcgg ctgtccatca ctgtccttca
     5821 ctatggcttt gatcccagga tgcagatcga gaagcacctg tcggcaccgt ccgcaggggc
     5881 tcaagatgcc cctgttctca tttccgatcg cgacgataca agtcaggttg ccagctgccg
     5941 cagcagcagc agtgcccagc accacgagtt ctgcacaagg tcccccagta aaatgatata
     6001 cattgacacc agtgaagatg cggccgtcgc tagagagagc tgcgctggcg acgctgtagt
     6061 cttcagagat ggggatgctg ttgattgtag ccgttgctct ttcaatgagg gtggattctt
     6121 cttgagacaa aggcttggcc atggggccgg ggttctcctc cacgtcgccg gcctgcttca
     6181 gcaggctgaa gttggtggcg ccgctgcccc cggggagcat gtcaaggtca aaatcgtcaa
     6241 gagcgtcagc aggcagcata tcaaggtcaa agtcgtcaag ggcatcggct gggagcatgt
     6301 ctaagtcaaa atcgtcaagg gcgtcggtcg gcccgccgct ttcgcacttt agctgtttct
     6361 ccaggccaca tatgattagt tccaggccga aaaggaaggc aggttcggct ccctgccggt
     6421 cgaacagctc aattgcttgt ttcagaagtg ggggcataga atcggtggta ggtgtctctc
     6481 tttcctcttt tgctacttga tgctcctgtt cctccaatac gcagcccagt gtaaagtggc
     6541 ccacggcgga cagagcgtac agtgcgttct ccagggagaa gccttgctga cacaggaacg
     6601 cgagctgatt ttccagggtt tcgtactgtt tctctgttgg gcgggtgccg agatgcactt
     6661 tagccccgtc gcgatgtgag aggagagcac agcggtatga cttggcgttg ttccgcagaa
     6721 agtcttgcca tgactcgcct tccagggggc aggagtgggt atgatgcctg tccagcatct
     6781 cgattggcag ggcatcgagc agggcccgct tgttcttcac gtgccagtac agggtaggct
     6841 gctcaactcc cagcttttga gcgagtttcc ttgtcgtcag gccttcgata ccgactccat
     6901 tgagtaattc cagagcagag tttatgactt tgctcttgtc cagtctagac atcttatcgt
     6961 catcgtcttt gtaatccatg gtggcggatc ccgcgtcacg acacctgtgt tctggcggca
     7021 aacccgttgc gaaaaagaac gttcacggcg actactgcac ttatatacgg ttctccccca
     7081 ccctcgggaa aaaggcggag ccagtacacg acatcacttt cccagtttac cccgcgccac
     7141 cttctctagg caccggttca attgccgacc cctcccccca acttctcggg gactgtgggc
     7201 gatgtgcgct ctgcccactg acgggcaccg gagccactcg agtggaatt
//
