LOCUS       sequence-309360-        7521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SB-transposon with constitutive bi-directional promoter. One side 
            contains Paullinia cupana ancestral Caffeine Synthase 2 (PcAncCS2); 
            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 7521)
  TITLE     Methylxanthine Synthesis in Human Cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7521)
  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            "Unpublished"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 PcAncCS2
FEATURES             Location/Qualifiers
     source          1..7521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1461..2162
                     /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(1594..1612)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1862..1881
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2056..2079
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2172..2228
                     /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             2229..2828
                     /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(2229..2248)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2725..2745
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2928..2947)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2993..3048
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3047..3066)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3440..3457)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3611..4199)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3691..3710)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4370..5161,5162..5230))
                     /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     4993..5012
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5231..5335)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5767..5991
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5980..5997
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     regulatory      7133..7142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(join(7168..7521,1..829))
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     complement(7200..7220)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 ccctgcagca gggccgggaa gcggccatct ttccgctcac gcaactggtg ccgaccgggc
       61 cagccttgcc gcccagggcg gggcgataca cggcggcgcg aggccaggca ccagagcagg
      121 ccggccagct tgagactacc cccgtccgat tctcggtggc cgcgctcgca ggccccgcct
      181 cgccgaacat gtgcgctggg acgcacgggc cccgtcgccg cccgcggccc caaaaaccga
      241 aataccagtg tgcagatctt ggcccgcatt tacaagacta tcttgccaga aaaaaagcgt
      301 cgcagcaggt catcaaaaat tttaaatggc tagagactta tcgaaagcag cgagacaggc
      361 gcgaaggtgc caccagattc gcacgcggcg gccccagcgc ccaagccagg cctcaactca
      421 agcacgaggc gaaggggctc cttaagcgca aggcctcgaa ctctcccacc cacttccaac
      481 ccgaagctcg ggatcaagaa tcacgtactg cagccagggg cgtggaagta attcaaggca
      541 cgcaagggcc ataacccgta aagaggccag gcccgcggga accacacacg gcacttacct
      601 gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact gcacttatat
      661 acggttctcc cccaccctcg ggaaaaaggc ggagccagta cacgacatca ctttcccagt
      721 ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc gacccctccc cccaacttct
      781 cggggactgt gggcgatgtg cgctctgccc actgacgggc accggagcct ctaggcgaga
      841 ccctgtctca caaaataaag taagcccgga ctgagtgcgg aaaggcgggc ctggcgggtc
      901 tggtctcccc atgcgggcca ccagaggccc tgcagccttc agtcgcttga aggggtaatg
      961 gcgcttccac tcacaaacat ggcggacaga gcgtgtgaac gagatgaaca gcccctcaaa
     1021 aatatggccg ccgaggctgg acggccgtgc cccagcagca ccgcctccgc gccccacgtg
     1081 atctctcgcc gggcacagcg ctgaccgcgg aggtccaacc ggaagaatgt ccggattgga
     1141 cattcggaag agggcccgcc ttccctgggg aatctctgcg cacgcgcaga acgcttcgac
     1201 caatgaaaac acaggaagcc gtccgcgcaa ccgcgttgcg tcacttctgc cgcccctgtt
     1261 tcaaggtata tagccgtaga cggaacttcg cctttctctc ggccttagcg ccattttttt
     1321 gggtgagtgt tttttggttc ctgcgttggg attccgtgta caatccatag acatctgacc
     1381 tcggcactta gcatcatcac agcaaactaa ctgtagcctt tctctctttc cctgtagaaa
     1441 cctctgcacc tgaggccacc atgttgagca agggcgagga ggtcatcaaa gagttcatgc
     1501 gcttcaaggt gcgcatggag ggctccatga acggccatga gttcgagatc gagggcgagg
     1561 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc
     1621 ccctgccctt cgcctgggac atcctgtccc cccagttcat gtacggctcc aaggcgtacg
     1681 tgaagcaccc cgccgacatc cccgattaca agaagctgtc cttccccgag ggcttcaagt
     1741 gggagcgcgt gatgaacttc gaggacggcg gtctggtgac cgtgacccag gactcctccc
     1801 tgcaggacgg cacgctgatc tacaaggtga agatgcgcgg caccaacttc ccccccgacg
     1861 gccccgtaat gcagaagaag acaatgggct gggaggcctc caccgagcgc ctgtaccccc
     1921 gcgacggcgt gctgaagggc gagatccacc aggccctgaa gctgaaggac ggcggccact
     1981 acctggtgga gttcaagacc atctacatgg ccaagaagcc cgtgcaactg cccggctact
     2041 actacgtgga caccaagctg gacatcacct cccacaacga ggactacacc atcgtggaac
     2101 agtacgagcg ctccgagggc cgccaccacc tgttcctgta cggcatggac gagctgtaca
     2161 agggcagtgg agctactaac ttcagcctgc tgaagcaggc tggtgacgtc gaggagaatc
     2221 ctggccccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     2281 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     2341 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     2401 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     2461 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     2521 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     2581 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     2641 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     2701 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     2761 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     2821 gtgcctgatt cgaaggcctg tcgtgaattc actcctcagg tgcaggctgc ctatcagaag
     2881 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct
     2941 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     3001 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     3061 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     3121 tatgccatat gctggctgcc atgaactagc tactcgggac cccttaccga aacatcgccg
     3181 cattctgcag aggagtcgag tgtatgtaaa cttctgaccc actgggaatg tgatgaaaga
     3241 aataaaagct gaaatgaatc attctctcta ctattattct gatatttcac attcttaaaa
     3301 taaagtggtg atcctaactg acctaagaca gggaattttt actaggatta aatgtcagga
     3361 attgtgaaaa agtgagttta aatgtatttg gctaaggtgt atgtaaactt ccgacttcaa
     3421 ctgtataggg atccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3481 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3541 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3601 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3661 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3721 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3781 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3841 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3901 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3961 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4021 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4081 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4141 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4201 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4261 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4321 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4381 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4441 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4501 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4561 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4621 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4681 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4741 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4801 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4861 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4921 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4981 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5041 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5101 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5161 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5221 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5281 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5341 ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     5401 taccgcatca ggaaattgta agcgttaata ttttgttaaa attggatccc tatacagttg
     5461 aagtcggaag tttacataca cttaagttgg agtcattaaa actcgttttt caactactcc
     5521 acaaatttct tgttaacaaa caatagtttt ggcaagtcag ttaggacatc tactttgtgc
     5581 atgacacaag tcatttttcc aacaattgtt tacagacaga ttatttcact tataattcac
     5641 tgtatcacaa ttccagtggg tcagaagttt acatacacta agttcgactc ctctgcagaa
     5701 tgcggcgatg tttcggtaag gggtccgcta tctagtaggc ccagctggtt ctttccgcct
     5761 cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc
     5821 ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg
     5881 atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg
     5941 aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca gtcgaggctg
     6001 atcagcgagc tctagagaat tgatccccaa gcttggcctg acaggccttc gaattaattc
     6061 cgcttcaggc ccacgacaaa gctgcactgt gtggcgaagt cctgcagtgg caggtcctgg
     6121 atctcgtcgt acagctcgtc caggatctga gggccgaagt ggtggctgat cattgtctcg
     6181 gtgatggatc tcaggttgtt gccgaagcct ctggcgtcgc tccaaatgcc ttcgtttctg
     6241 gcgccgatcc ggatggtgaa ggtttccatc agttccacgg cgtagctgcc ttcgccttcg
     6301 accagctctt tcacttcttg agcgctaggg gtgtagtagg gcacctcgaa gctgtccagc
     6361 ttttcctcgt cgatcagtcc cttagacacc agtctgctga tggcctgaga caggatcagc
     6421 caaggcagct gcagttcttt ctcgcagctg aaatcctggc tcagccttcc atgcagcacc
     6481 agcaccactc tgccgttgga caccatttcc tcggctctgg acttcagaaa cagggtgaag
     6541 tccttctgga actgggccag ataagcttcg cgcacggcag aggggcttgt cttgctgatg
     6601 tagaaggtgc ccttgttgat ggggttgccc ttctcatctc tcaggtcggg caccttgctc
     6661 agccagtgca cgctgtagtt gctgtacacc aggtggatgg tgttcagagg gaacagtctg
     6721 ccgtgaaagc tgccaggggc tcccatgacc agacagctgg tgttctcgaa ttcctcgccg
     6781 ccagagccaa caaagccgct caggcccttg aacagggtgt tgaagtcgtt gctaggcagg
     6841 tcgttcaggt agaactggaa ctcggggatc ttgtggttca gctctctgca cttgttgacc
     6901 acggtctgca ccacggtgct catgatggtg ttgcttgtgc tgccagaggc acagcccaga
     6961 tcggccacgt tcagcagatg ctcgaatgga gggctttcct ctttgaacag gctctcgatg
     7021 gctctcttca gtgtggggat agacttcacg gcggtgatgt tggtgaactt gctgttcagc
     7081 aggtagctgc tctcgccctc gcctttgttc atgcacagca cgtccttcac gtccatggtg
     7141 gcctcagagg ccagcttggg gtagttttca cgacacctta aatggaagaa aaaaactttg
     7201 aaccactgtc tgaggcttga gaatgaacca agatccaaac tcaaaaaggg caaattccaa
     7261 ggagaattac atcaagtgcc aagctggcct aacttcagtc tccacccact cagtatgggg
     7321 aaactccatc gcataaaacc cctcccccca acctaaagac gacgtactcc aaaagctcga
     7381 gaactaatcg aggtgcctgg acggcgcccg gtactccgtg gagtcacatg aagcgacggc
     7441 tgaggacgga aaggcccttt tcctttgtgt gggtgactca cccgcccgct ctcccgagcg
     7501 ccgcgtcctc cattttgagc t
//
