LOCUS       sequence-309363-        7272 bp    DNA     circular SYN 15-APR-2025
DEFINITION  SB-transposon with inducible expression of dTomato and Coffea 
            arabica monomethylxathine methyltransferase via P2A self-cleaving 
            linker in a Tet-inducible pSBTRE(-Delta-rtTA)-GB backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7272)
  TITLE     Methylxanthine Synthesis in Human Cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7272)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pSBTet(-Delta-rtTA)-GB dTomato-P2A-CaMXMT
FEATURES             Location/Qualifiers
     source          1..7272
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(635..637,638..640,641..1351)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MLSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(635..637,638..640,641..1351)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MLSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(680..701)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(941..960)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1288..1309
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1361..1417
                     /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             1418..1816
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAMPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(1916..1935)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1981..2036
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2035..2054)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2428..2445)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2599..3187)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2679..2698)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3358..4149,4150..4218))
                     /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     3981..4000
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4219..4323)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4726..5038
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    4734..4752
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4770..4788
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4805..4823
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4841..4859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4877..4895
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4912..4930
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4948..4966
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5011..5035
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5117..5818
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MLSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFTPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(5250..5268)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5518..5537
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5828..5884
                     /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"
     polyA_signal    7063..7184
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7074..7093)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7128..7147
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 gtacctaggc gagaccctgt ctcacaaaat aaagtaagcc cggactgagt gcggaaaggc
       61 gggcctggcg ggtctggtct ccccatgcgg gccaccagag gccctgcagc cttcagtcgc
      121 ttgaaggggt aatggcgctt ccactcacaa acatggcgga cagagcgtgt gaacgagatg
      181 aacagcccct caaaaatatg gccgccgagg ctggacggcc gtgccccagc agcaccgcct
      241 ccgcgcccca cgtgatctct cgccgggcac agcgctgacc gcggaggtcc aaccggaaga
      301 atgtccggat tggacattcg gaagagggcc cgccttccct ggggaatctc tgcgcacgcg
      361 cagaacgctt cgaccaatga aaacacagga agccgtccgc gcaaccgcgt tgcgtcactt
      421 ctgccgcccc tgtttcaagg tatatagccg tagacggaac ttcgcctttc tctcggcctt
      481 agcgccattt ttttgggtga gtgttttttg gttcctgcgt tgggattccg tgtacaatcc
      541 atagacatct gacctcggca cttagcatca tcacagcaaa ctaactgtag cctttctctc
      601 tttccctgta gaaacctctg cacctgaggc caccatgttg agcaagggcg aggagctgtt
      661 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
      721 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
      781 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
      841 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
      901 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
      961 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1021 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1081 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1141 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1201 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     1261 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     1321 gatcactctc ggcatggacg agctgtacaa gggcagtgga gctactaact tcagcctgct
     1381 gaagcaggct ggtgacgtcg aggagaatcc tggccccatg gccatgcctt tgtctcaaga
     1441 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
     1501 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
     1561 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
     1621 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
     1681 cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
     1741 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
     1801 tgtgtgggag ggctaattcg aaggcctgtc gtgaattcac tcctcaggtg caggctgcct
     1861 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     1921 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     1981 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     2041 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     2101 tggcaacata tgccatatgc tggctgccat gaactagcta ctcgggaccc cttaccgaaa
     2161 catcgccgca ttctgcagag gagtcgagtg tatgtaaact tctgacccac tgggaatgtg
     2221 atgaaagaaa taaaagctga aatgaatcat tctctctact attattctga tatttcacat
     2281 tcttaaaata aagtggtgat cctaactgac ctaagacagg gaatttttac taggattaaa
     2341 tgtcaggaat tgtgaaaaag tgagtttaaa tgtatttggc taaggtgtat gtaaacttcc
     2401 gacttcaact gtatagggat ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2461 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2521 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2581 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2641 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2701 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2761 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2821 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2881 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2941 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3001 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3061 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3121 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3181 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3241 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3301 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3361 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3421 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3481 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     3541 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3601 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3661 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3721 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3781 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3841 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3901 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3961 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4021 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4081 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4141 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4201 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4261 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4321 gcgcacattt ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa
     4381 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tggatcccta
     4441 tacagttgaa gtcggaagtt tacatacact taagttggag tcattaaaac tcgtttttca
     4501 actactccac aaatttcttg ttaacaaaca atagttttgg caagtcagtt aggacatcta
     4561 ctttgtgcat gacacaagtc atttttccaa caattgttta cagacagatt atttcactta
     4621 taattcactg tatcacaatt ccagtgggtc agaagtttac atacactaag ttcgactcct
     4681 ctgcagaatg cggcgatgtt tcggtaaggg gtccgctatc tagacgagtt tactccctat
     4741 cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac gatgtcgagt
     4801 ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa
     4861 cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt atccctatca
     4921 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgatta
     4981 ggcgtgtacg gtgggcgcct ataaaagcag agctcgttta gtgaaccgtc agatcgcctg
     5041 gagccaattc caactctttt gccttatacc tactttccgt accacttcct accctcgaaa
     5101 ggcctctgag gccaccatgt tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt
     5161 caaggtgcgc atggagggct ccatgaacgg ccatgagttc gagatcgagg gcgagggcga
     5221 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct
     5281 gcccttcgcc tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa
     5341 gcaccccgcc gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga
     5401 gcgcgtgatg aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca
     5461 ggacggcacg ctgatctaca aggtgaagat gcgcggcacc aacttcaccc ccgacggccc
     5521 cgtaatgcag aagaagacaa tgggctggga ggcctccacc gagcgcctgt acccccgcga
     5581 cggcgtgctg aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccactacct
     5641 ggtggagttc aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta
     5701 cgtggacacc aagctggaca tcacctcaca caacgaggac tacaccatcg tggaacagta
     5761 cgagcgctcc gagggccgcc accacctgtt cctgtacggc atggacgagc tgtacaaggg
     5821 cagtggagct actaacttca gcctgctgaa gcaggctggt gacgtcgagg agaatcctgg
     5881 ccccatggaa ctgcaagagg tgctgcacat gaacgaaggc gagggcgata ccagctacgc
     5941 caagaacgcc agctacaatc tggccctggc caaagtgaag cccttcctgg aacagtgcat
     6001 cagagagctg ctgcgggcca acctgcctaa catcaacaag tgcatcaagg tggccgacct
     6061 gggctgtgcc tctggaccta atactctgct gaccgtgcgg gacatcgtgc agagcatcga
     6121 caaagtgggc caagaggaaa agaacgagct ggaacggcct accatccaga tctttctgaa
     6181 cgacctgttc cagaacgact tcaacagcgt gttcaagctg ctgcccagct tctaccggaa
     6241 gctggaaaaa gagaacggcc ggaagatcgg ctcctgcctg atttctgcca tgcctggcag
     6301 cttttacggc cggctgttcc ccgaggaatc catgcacttt ctgcacagct gctacagcgt
     6361 gcactggctg tctcaggtgc catctggcct ggttatcgaa ctcggcatcg gagccaacaa
     6421 gggcagcatc tacagcagca agggctgcag acctccagtg cagaaggcct acctggacca
     6481 gttcaccaag gacttcacca cctttctgcg gatccacagc aaagagctgt tcagccgggg
     6541 cagaatgctg ctgacatgca tctgcaaggt ggacgagttc gacgagccca atccactgga
     6601 cctgctggac atggccatca acgacctgat cgtggaaggc ctgctggaag aagaaaagct
     6661 ggacagcttc aacatcccgt tcttcacccc tagcgccgag gaagtgaagt gcattgtgga
     6721 agaggaaggc agctgcgaga tcctgtacct ggaaaccttc aaggcccact acgacgccgc
     6781 cttcagcatc gacgatgatt accctgtgcg gagccacgag cagatcaagg ccgaatatgt
     6841 ggccagcctg atcagaagcg tgtacgagcc tatcctggcc agccactttg gcgaggccat
     6901 catgcctgat ctgttccaca gactggccaa gcacgccgct aaagtgctgc atatgggcaa
     6961 gggatgctac aacaacctga tcatcagcct ggccaagaag cccgagaagt ccgacgtgta
     7021 attcgaaggc ctgtcaggcc aagcttccat cgatagacat gataagatac attgatgagt
     7081 ttggacaaac cacaacaaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     7141 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     7201 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc
     7261 tctacaaatg tg
//
