LOCUS       sequence-309353-        7562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SB-transposon with constitutive bi-directional promoter. One side 
            contains coffee caffeine synthase 1 (CCS1) CCS-CTS deletion mutant; 
            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 7562)
  TITLE     Methylxanthine Synthesis in Human Cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7562)
  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            "Unpublished"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 CCS1(Delta-304-316)
FEATURES             Location/Qualifiers
     source          1..7562
                     /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"
     regulatory      5823..5832
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(7009..7026)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7015..7239
                     /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 aattacttcc
     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 ggaactgcaa gaggtgctgc acatgaatgg cggcgagggc gatacaagct
     5881 acgccaagaa cagcagctac aacctgttcc tgatcagagt gaagcccgtg ctggaacagt
     5941 gcatccaaga gctgctgaga gccaacctgc ctaacatcaa caagtgcttc aaagtgggcg
     6001 acctgggctg tgccagcgga cctaatacct ttagcaccgt gcgggacatc gtgcagagca
     6061 tcgacaaagt gggacaagag aagaagaacg agctggaacg gcctaccatc cagatctttc
     6121 tgaacgacct gttccagaac gacttcaaca gcgtgttcaa gctgctgccc agcttctacc
     6181 ggaacctgga aaaagagaac ggccggaaga tcggctcctg cctgattgga gctatgcccg
     6241 gctccttcta cagcagactg ttccccgagg aatccatgca ctttctgcac agctgctact
     6301 gcctgcactg gctgtctcag gtgccatctg gcctggttac agagctgggc atctctgcca
     6361 acaagggctg catctacagc tccaaggcca gcgggcctcc tatcaagaag gcctacctgg
     6421 accagttcac caaggacttc accacctttc tgcggatcca cagcgaggaa ctgatcagca
     6481 gaggccggat gctgctgacc ttcatctgca aagaggacga gttcgatcac cccaacagca
     6541 tggacctgct ggaaatgagc atcaacgacc tggtcatcga gggccacctg gaagaggaaa
     6601 agctggacag cttcaacgtg cccatctacg cccctagcac cgaggaagtg aagcggatcg
     6661 ttgaggaaga gggcagcttc gagatcctgt acctggaaac cttttacgcc ccttacgacg
     6721 ccggcttcag catcgacgat gagcacgcca gagctgctca cgtggcatct gttgtgcgga
     6781 gcatctacga gcctatcctg gcctctcact tcggcgaggc cattctgcct gatctgagcc
     6841 accggatcgc caagaatgcc gccaaggtgc tgagaagcgg caagggcttt tacgacagcg
     6901 tgatcatcag cctggccaag aagcccgaga aggccgatat gtagggtaac tattcgaagg
     6961 cctgtcaggc caagcttggg gatcaattct ctagagctcg ctgatcagcc tcgactgtgc
     7021 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     7081 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     7141 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     7201 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     7261 gctgggccta ctagatagcg gaccccttac cgaaacatcg ccgcattctg cagaggagtc
     7321 gaacttagtg tatgtaaact tctgacccac tggaattgtg atacagtgaa ttataagtga
     7381 aataatctgt ctgtaaacaa ttgttggaaa aatgacttgt gtcatgcaca aagtagatgt
     7441 cctaactgac ttgccaaaac tattgtttgt taacaagaaa tttgtggagt agttgaaaaa
     7501 cgagttttaa tgactccaac ttaagtgtat gtaaacttcc gacttcaact gtatagggat
     7561 cc
//
