LOCUS       sequence-309354-        8309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SB-transposon with constitutive bi-directional promoter. One side 
            contains N-GST coffee caffeine synthase 1 (GST-CCS1) fusion; the 
            other side contains RFP and puromycin resistance genes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8309)
  TITLE     Methylxanthine Synthesis in Human Cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8309)
  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            "Unpublished"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: pSBBi-RP GST-CCS1
FEATURES             Location/Qualifiers
     source          1..8309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        109..213
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(214..282,283..1074)
                     /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(432..451)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1245..1833
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1734..1753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1987..2004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2378..2397
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    2396..2451
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     2497..2516
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(2616..3215)
                     /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(2699..2719)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3196..3215
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(3216..3272)
                     /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(3282..3983)
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MLSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(3365..3388)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3563..3582)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3832..3850
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     promoter        4615..5797
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     5745..5765
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5829..6482
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     6440..6462
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             6501..6521
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(7756..7773)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7762..7986
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
       61 tgtgcggtat ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct
      121 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
      181 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
      241 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
      301 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
      361 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
      421 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
      481 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
      541 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
      601 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
      661 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
      721 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
      781 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
      841 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
      901 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
      961 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     1021 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     1081 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     1141 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     1201 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1261 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1321 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1381 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1441 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1501 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1561 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1621 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1681 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     1741 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     1801 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     1861 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     1921 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     1981 gagcgcagcg agtcagtgag cgaggaagcg gatccctata cagttgaagt cggaagttta
     2041 catacacctt agccaaatac atttaaactc actttttcac aattcctgac atttaatcct
     2101 agtaaaaatt ccctgtctta ggtcagttag gatcaccact ttattttaag aatgtgaaat
     2161 atcagaataa tagtagagag aatgattcat ttcagctttt atttctttca tcacattccc
     2221 agtgggtcag aagtttacat acactcgact cctctgcaga atgcggcgat gtttcggtaa
     2281 ggggtcccga gtagctagtt catggcagcc agcatatggc atatgttgcc aaactctaaa
     2341 ccaaatactc attctgatgt tttaaatgat ttgccctccc atatgtcctt ccgagtgaga
     2401 gacacaaaaa attccaacac actattgcaa tgaaaataaa tttcctttat tagccagaag
     2461 tcagatgctc aaggggcttc atgatgtccc cataattttt ggcagaggga aaaagatctc
     2521 agtggtattt gtgagccagg gcattggcca caccagccac caccttctga taggcagcct
     2581 gcacctgagg agtgaattca cgacaggcct tcgaatcagg caccgggctt gcgggtcatg
     2641 caccaggtgc gcggtccttc gggcacctcg acgtcggcgg tgacggtgaa gccgagccgc
     2701 tcgtagaagg ggaggttgcg gggcgcggag gtctccagga aggcgggcac cccggcgcgc
     2761 tcggccgcct ccactccggg gagcacgacg gcgctgccca gacccttgcc ctggtggtcg
     2821 ggcgagacgc cgacggtggc caggaaccac gcgggctcct tgggccggtg cggcgccagg
     2881 aggccttcca tctgttgctg cgcggccagc cgggaaccgc tcaactcggc catgcgcggg
     2941 ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg gcgtggtcca gaccgccacc
     3001 gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga gcccgacgcg cgtgaggaag
     3061 agttcttgca gctcggtgac ccgctcgatg tggcggtccg gatcgacggt gtggcgcgtg
     3121 gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg ccctggggac gtcgtcgcgg
     3181 gtggcgaggc gcaccgtggg cttgtactcg gtcatggggc caggattctc ctcgacgtca
     3241 ccagcctgct tcagcaggct gaagttagta gctccactgc ccttgtacag ctcgtccatg
     3301 ccgtacagga acaggtggtg gcggccctcg gagcgctcgt actgttccac gatggtgtag
     3361 tcctcgttgt gggaggtgat gtccagcttg gtgtccacgt agtagtagcc gggcagttgc
     3421 acgggcttct tggccatgta gatggtcttg aactccacca ggtagtggcc gccgtccttc
     3481 agcttcaggg cctggtggat ctcgcccttc agcacgccgt cgcgggggta caggcgctcg
     3541 gtggaggcct cccagcccat tgtcttcttc tgcattacgg ggccgtcggg ggggaagttg
     3601 gtgccgcgca tcttcacctt gtagatcagc gtgccgtcct gcagggagga gtcctgggtc
     3661 acggtcacca gaccgccgtc ctcgaagttc atcacgcgct cccacttgaa gccctcgggg
     3721 aaggacagct tcttgtaatc ggggatgtcg gcggggtgct tcacgtacgc cttggagccg
     3781 tacatgaact ggggggacag gatgtcccag gcgaagggca gggggccgcc cttggtcacc
     3841 ttcagcttgg cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg
     3901 aactcatggc cgttcatgga gccctccatg cgcaccttga agcgcatgaa ctctttgatg
     3961 acctcctcgc ccttgctcaa catggtggcc tcaggtgcag aggtttctac agggaaagag
     4021 agaaaggcta cagttagttt gctgtgatga tgctaagtgc cgaggtcaga tgtctatgga
     4081 ttgtacacgg aatcccaacg caggaaccaa aaaacactca cccaaaaaaa tggcgctaag
     4141 gccgagagaa aggcgaagtt ccgtctacgg ctatatacct tgaaacaggg gcggcagaag
     4201 tgacgcaacg cggttgcgcg gacggcttcc tgtgttttca ttggtcgaag cgttctgcgc
     4261 gtgcgcagag attccccagg gaaggcgggc cctcttccga atgtccaatc cggacattct
     4321 tccggttgga cctccgcggt cagcgctgtg cccggcgaga gatcacgtgg ggcgcggagg
     4381 cggtgctgct ggggcacggc cgtccagcct cggcggccat atttttgagg ggctgttcat
     4441 ctcgttcaca cgctctgtcc gccatgtttg tgagtggaag cgccattacc ccttcaagcg
     4501 actgaaggct gcagggcctc tggtggcccg catggggaga ccagacccgc caggcccgcc
     4561 tttccgcact cagtccgggc ttactttatt ttgtgagaca gggtctcgcc tagaggctcc
     4621 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     4681 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     4741 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     4801 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt
     4861 ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacctcc
     4921 acgcccctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga
     4981 gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggct
     5041 tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt
     5101 cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca
     5161 agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc
     5221 gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc
     5281 gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg
     5341 cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt
     5401 tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac
     5461 gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
     5521 ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta
     5581 gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga
     5641 gtttccccat actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt
     5701 ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt
     5761 ggttcaaagt ttttttcttc catttaaggt gtcgtgaaaa ctaccccaag ctggcctctg
     5821 aggccaccat gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc
     5881 gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag
     5941 gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt
     6001 atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca
     6061 agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag
     6121 cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc
     6181 tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat
     6241 gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg
     6301 ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt
     6361 gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt
     6421 atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa
     6481 aatcggatag cagcggcaga gaaaacttgt atttccaggg cagagaccct gaaagatccg
     6541 aactgcaaga ggtgctgcac atgaatggcg gcgagggcga tacaagctac gccaagaaca
     6601 gcagctacaa cctgttcctg atcagagtga agcccgtgct ggaacagtgc atccaagagc
     6661 tgctgagagc caacctgcct aacatcaaca agtgcttcaa agtgggcgac ctgggctgtg
     6721 ccagcggacc taataccttt agcaccgtgc gggacatcgt gcagagcatc gacaaagtgg
     6781 gacaagagaa gaagaacgag ctggaacggc ctaccatcca gatctttctg aacgacctgt
     6841 tccagaacga cttcaacagc gtgttcaagc tgctgcccag cttctaccgg aacctggaaa
     6901 aagagaacgg ccggaagatc ggctcctgcc tgattggagc tatgcccggc tccttctaca
     6961 gcagactgtt ccccgaggaa tccatgcact ttctgcacag ctgctactgc ctgcactggc
     7021 tgtctcaggt gccatctggc ctggttacag agctgggcat ctctgccaac aagggctgca
     7081 tctacagctc caaggccagc gggcctccta tcaagaaggc ctacctggac cagttcacca
     7141 aggacttcac cacctttctg cggatccaca gcgaggaact gatcagcaga ggccggatgc
     7201 tgctgacctt catctgcaaa gaggacgagt tcgatcaccc caacagcatg gacctgctgg
     7261 aaatgagcat caacgacctg gtcatcgagg gccacctgga agaggaaaag ctggacagct
     7321 tcaacgtgcc catctacgcc cctagcaccg aggaagtgaa gcggatcgtt gaggaagagg
     7381 gcagcttcga gatcctgtac ctggaaacct tttacgcccc ttacgacgcc ggcttcagca
     7441 tcgacgatga ctaccagggc agatctcaca gccctgtgtc ctgtgatgag cacgccagag
     7501 ctgctcacgt ggcatctgtt gtgcggagca tctacgagcc tatcctggcc tctcacttcg
     7561 gcgaggccat tctgcctgat ctgagccacc ggatcgccaa gaatgccgcc aaggtgctga
     7621 gaagcggcaa gggcttttac gacagcgtga tcatcagcct ggccaagaag cccgagaagg
     7681 ccgatatgta gggtaactat tcgaaggcct gtcaggccaa gcttggggat caattctcta
     7741 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     7801 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     7861 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     7921 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     7981 ctatggcttc tgaggcggaa agaaccagct gggcctacta gatagcggac cccttaccga
     8041 aacatcgccg cattctgcag aggagtcgaa cttagtgtat gtaaacttct gacccactgg
     8101 aattgtgata cagtgaatta taagtgaaat aatctgtctg taaacaattg ttggaaaaat
     8161 gacttgtgtc atgcacaaag tagatgtcct aactgacttg ccaaaactat tgtttgttaa
     8221 caagaaattt gtggagtagt tgaaaaacga gttttaatga ctccaactta agtgtatgta
     8281 aacttccgac ttcaactgta tagggatcc
//
