LOCUS       sequence-134450-        8920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-demethylases for the degredation of caffeine and related
            methylxanthines. Can be used to measure caffeine content as 
            described in the publication listed below..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8920)
  AUTHORS   Quandt EM, Hammerling MJ, Summers RM, Otoupal PB, Slater B, Alnahhas
            RN, Dasgupta A, Bachman JL, Subramanian MV, Barrick JE
  TITLE     Decaffeination and measurement of caffeine content by addicted 
            Escherichia coli with a refactored N-demethylation operon from 
            Pseudomonas putida CBB5.
  JOURNAL   ACS Synth Biol. 2013 Jun 21;2(6):301-7. doi: 10.1021/sb4000146. Epub
            2013 Mar 22.
  PUBMED    23654268
REFERENCE   2  (bases 1 to 8920)
  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"; doi: "10.1021/sb4000146";
            journalName: "ACS Synth Biol"; date: "2013-06-21- 21"; volume: "2"; 
            issue: "6"; pages: "301-7"
COMMENT     Sequence Label: pDCAF3-IS5
FEATURES             Location/Qualifiers
     source          1..8920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     221..239
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(243..286)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    289..310
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     RBS             1562..1570
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     RBS             2641..2652
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             3744..3755
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             4624..4635
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             6453..6464
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     misc_feature    7161..7181
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      7182..7239
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(7263..7280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7434..8022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7514..7533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(8205..8299)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(8320..8920,1..59))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     8894..8913
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt
       61 ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt
      121 atttcattat ggtgaaagtt ggaacctctt acgtgcccga tcaactcgag tgccacctga
      181 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggca
      241 gaatttcaga taaaaaaaat ccttagcttt cgctaaggat gatttctgga attcgcggcc
      301 gcttctagag ttgacggcta gctcagtcct agggaaggtg cgaacaagtc cctgatatga
      361 gatcatgttt gtcatctgga gccatagaac agggttcatc atgagtcatc aacttacctt
      421 cgccgacagt gaattcagca gtaagcgccg tcagaccaga aaagagattt tcttgtcccg
      481 catggagcag attctgccat ggcaaaacat ggtggaagtc atcgagccgt tttaccccaa
      541 ggctggtaat ggccggcgac cttatccgct ggaaaccatg ctacgcattc actgcatgca
      601 gcattggtac aacctgagcg atggcgcgat ggaagatgct ctgtacgaaa tcgcctccat
      661 gcgtctgttt gcccggttat ccctggatag cgccttgccg gaccgcacca ccatcatgaa
      721 tttccgccac ctgctggagc agcatcaact ggcccgccaa ttgttcaaga ccatcaatcg
      781 ctggctggcc gaagcaggcg tcatgatgac tcaaggcacc ttggtcgatg ccaccatcat
      841 tgaggcaccc agctcgacca agaacaaaga gcagcaacgc gatccggaga tgcatcagac
      901 caagaaaggc aatcagtggc actttggcat gaaggcccac attggtgtcg atgccaagag
      961 tggcctgacc cacagcctgg tcaccaccgc ggccaacgag catgacctca atcagctggg
     1021 taatctgctg catggagagg agcaatttgt ctcagccgat gccggctacc aaggggcgcc
     1081 acagcgcgag gagctggccg aggtggatgt ggactggctg atcgccgagc gccccggcaa
     1141 ggtaagaacc ttgaaacagc atccacgcaa gaacaaaacg gccatcaaca tcgaatacat
     1201 gaaagccagc atccgggcca gggtggagca cccatttcgc atcatcaagc gacagttcgg
     1261 cttcgtgaaa gccagataca aggggttgct gaaaaacgat aaccaactgg cgatgttatt
     1321 cacgctggcc aacctgtttc gggcggacca aatgatacgt cagtgggaga gatctcacta
     1381 aaaactgggg ataacgcctt aaatggcgaa gaaacggtct aaataggctg attcaaggca
     1441 tttacgggag aaaaaatcgg ctcaaacatg aagaaatgaa atgactgagt cagccgagaa
     1501 gaatttcccc gcttattcgc accttcccta ggtacagtgc tagctactag agaaatcaaa
     1561 ttaaggaggt aagataaatg gaacaggcaa tcattaatga tgaacgggag tatcttcgcc
     1621 atttctggca tcccgtatgt actgtaactg agcttgaaaa ggcgcatcct tccagcctcg
     1681 gccccctggc cgttaagctg ctgaatgaac agctcgttgt cgccaagcta ggcgatgagt
     1741 acgtcgcgat gcgtgataga tgcgctcatc gatcagctaa gctttccttg ggtacagtaa
     1801 gtggaaaccg cctacagtgc ccctatcacg gatggcaata tgatacgcat ggcgcttgcc
     1861 agctcgtacc agcgtgcccc aacagcccaa tacccaacaa ggctaaagtt gatcgcttcg
     1921 attgcgaaga acgctatgga ttgatttgga ttcgattgga ctctagtttt gactgcactg
     1981 aaattcccta ctttagtgca gccaacgatc ctaggttgcg tattgttata caagaacctt
     2041 actggtggga tgcgactgca gaacgtagat gggaaaattt tacagatttt tctcactttg
     2101 cattcattca cccaggcacg cttttcgatc caaataatgc tgaacctcca attgttccga
     2161 tggatcgatt caatggtcag tttcggtttg tctacgacac gccagaagat atggctgtcc
     2221 caaatcaggc tccaattggt tcattttcgt atacttgcag catgccgttt gctattaacc
     2281 ttgaagtatc caaatactcc agcagttcgc tgcatgtgtt attcaatgtg tcatgtccgg
     2341 tagacagcca caccacgaaa aactttctga tcttcgctag ggagcaatcg gacgactcgg
     2401 attatctgca cattgcattt aatgatctcg tcttcgctga agacaaacca gtaattgagt
     2461 cccaatggcc taaagacgcg ccagcagatg aggtctcagt agtcgcagat aaggtatcga
     2521 tacaatatag aaaatggctg cgggaactaa aagaagctca taaagaaggt tcacaagcct
     2581 tccgaagtgc tttgttagac ccagtcattg aaagcgatag gagctacata taatctagag
     2641 aaagaggaga aatactagat gaaagaacag ctcaagccgc tgctagaaga caagacttac
     2701 cttcgccact tctggcatcc cgtgtgtacc cttaatgaat tcgaacgcgc caacgccagt
     2761 gggcacggcc ccatgggcgt caccttgcta ggcgagaaat tggtgttggc caggttaaat
     2821 tcaaagatca ttgcggctgc tgaccgatgt gctcatcgat cggcacagct ctccatcggc
     2881 cgcgtttgca gcaacgccgg aaaggactat ctcgaatgcc cgtatcacgg ctggcgctac
     2941 gatgaggctg gggcctgtca actgatccct gcttgccctg acaaaagcat ctctcctcgg
     3001 gccaagattt cctcattcga ttgtgaggtg aaatacgaca tcgtgtgggt acggctggat
     3061 aacagcttcg attgcactca gattccatac ctcagcgatt tcgataatcc cgacatgcag
     3121 gtaatcgttg ccgattcgta tatttgggag actgttgccg agcggcggtg ggagaacttt
     3181 acagattttt cgcactttgc cttcgtacac ccagggacgc tctatgatcc gtttttcgct
     3241 agccacccaa ctgtttacgt gaatcgcgtt gatggtgagt tgcaattcaa actcgctccg
     3301 ccgcgtgaaa tgaaaggcat cccgccagaa gcaccgatgg gtgacttcac ctaccgctgc
     3361 acaatgccgt attcagtaaa tcttgaaatc aaattgtgga aagatgactc acgtttcgtt
     3421 ctttggacta ccgctagccc tgtcgataac aagtcttgcc ggaattttat gattattgtg
     3481 cgtgagaagg ataatcaacc tgatcatatg cacctggctt tccagaagcg ggtgcttgac
     3541 gaagaccagc ctgttatcga atcgcaatgg cctctcgaaa tacagacctc ggaagtctcc
     3601 gttgcaaccg ataaaatttc cgtccagttc cgcaaatggc ataaagagct atctctgtca
     3661 gccgttgaag gacgggaggc gttccgcgat tccgtcttga cgaatgttat tgaagaagaa
     3721 cagtaagcga gaccggtcta gagaaagagg agaaatacta gatgtctact gaccaagtaa
     3781 tttttaacga ctggcatcca gtcgctgctt tggaagatgt atctctcgat aaacgatacc
     3841 gctgtcgact actaggtcgt acagttagtt atgtgaaaac atctgatgct gttaatgctc
     3901 attgggaaga aagtgcagat gaaattaaga ccatccgcgc taaagaaatc tatggtcttc
     3961 tgtggctctc ctttgccgac aaacccagtg agatgttcga tattgcagag ttcaaggaac
     4021 ctgatcgccg aatcgtcagc gctggatctg tgcgcgtaaa tgtctcagga ctgcgtgcta
     4081 tcgaaaactt tctggacatg gctcatttcc cttttgttca tacggatatt ttgggtgcag
     4141 agccactgac ggaagttgag ccgtataacg tcaactatga tgaaactgtt gatgagatct
     4201 tcgctactga gtgtaagttc ccgcaaccta aaggctcagc tactgctgtc gagccaattg
     4261 atatgcagta tatataccgc attacacggc catattccgc cattttatat aaaacttgcc
     4321 cgcccgaacc gcatagatgg gatgccctcg gtctgtttat tcaacctgta gacgaagatt
     4381 ggtgcatagc gcatacaatt atgtgctatg tggatgacgt taactcagat caacaacttc
     4441 gccatttcca gcagacgatt tttggccagg acttgatgat tcttatcaac caagttccaa
     4501 agcgtcttcc cctggctgca agtcgagaga gcccagtccg ggccgatgtg cttgcaacag
     4561 cttatcgtcg ctggctgcgt gagaaaggtg tgcaatatgg tgctctgcgg gactaatcta
     4621 gagaaagagg agaaatacta gatgaacaaa cttgacgtca accagtggtt tcctattgct
     4681 accactgaag atctcccgaa gcgccatgtc tttcatgcca cgttgttggg gcaagaaatg
     4741 gccatctggc gcgatgactc tggttcagtt aatgcttggg agaaccgctg cccgcataga
     4801 ggattgcggt tgacactggg tgctaatacc ggtaacgagt tgcggtgtca gtatcatgga
     4861 tggacttatg aaagcgggac tggtggctgc acttttgtcc cagcccatcg cgatgcacca
     4921 cccccaaatg ccgcgcgggt taatactttt cctgtccgcg aaaagcacgg ctttatctgg
     4981 acgacattag gtcagccgcc aggagagccc atttcaatcc tcgatgacgc tcagcttgta
     5041 aacgctgtaa aaacaaatct gcatagcgta gttatagatg ctgatattga cggagttgtc
     5101 agcgtcctac gtcagaatct ttcagcgttc atcgatgtgt ttggtgcggc cagcgctgaa
     5161 gatctgcatt tgaaatccat gctgcaagat cgagggattc tggtaacaag atcaggctct
     5221 attgctattc atttttatat gcagcgctca accattagta aatgcgttgt acatgcgcaa
     5281 gtacttactc cgggacgtcc aggatacgaa cttcaaaaga actactcgta tgccatgaac
     5341 gttatccgca gggcagcaga agctgtagct accgacttga ttagcattac agatatcagc
     5401 gatcagacta tcgaaaagct tgaagtcgtt agagaaaaca tgactaaggc tcctccaacc
     5461 cactatatct gcgaagtggt tacgcgtact caagagacag gtgatattaa ctcatactgg
     5521 ctgaagccta tcggctaccc actaccagca ttcagtccag ggatgcacat cagcatcaca
     5581 acgccggagg gtagcattcg acaatattcc ctcgtgaacg ggcctgacga gcgtgaatcc
     5641 ttcatcatcg gtgtgaagaa agagattcag tcccgtggcg gctccagatc aatgcacgaa
     5701 gatgtgaagg ttggaacgca actaaaagtt acacttccga ggaacggttt tccactcgtc
     5761 caaaccagaa aacacccgat tctcgtagca ggtggcatcg gtatcacccc aattttgtgt
     5821 atggcacagg ctctggatca gcaaggttca tcgtatgaaa tacattattt tgctcgtgca
     5881 tttgagcatg ttccattcca ggatcgactg actgcgttgg gcgatcgttt gaatgtgcat
     5941 cttggcctcg gcccagacga gactagagca aaacttgccg acatcatgga gattcataac
     6001 gcccaagacg tagatgttta cacttgcggc ccgcaaccaa tgatcgaaac tgtatctgct
     6061 gtcgctcttg ctcatggcat cgctgaagag tccatccgat ttgaattttt cagtaaaaag
     6121 aacgatgttc ccgtttctga tgaagaatat gaggttgagc tcaaaaaaac tggtcaaata
     6181 ttcactgtct cgcctggctc tacgttgttg caagcttgtt tggacaacga tgttcgtatc
     6241 gaagcttctt gtgagcaggg tgtatgcggg acttgtataa ctccagtcgt atccggcgat
     6301 ctcgagcatc atgacactta cctttctaag aaagaaaggg aaagcggtaa gtggatcatg
     6361 ccgtgtgttt cgcgctgcaa gtccaaaaaa atcgttctcg atctgtgagg ccgggcatca
     6421 cccacggtat ggacgagctg tataaactag agaaagagga gaaatactag atgatcacgc
     6481 tttatgacta tgaactctcg gggaattgct acaaggtgcg gatgctcctt tccatcctga
     6541 agctaccgta cacgatagag acagtggaat ttttcccgtc cagggaacat aaatcaccgg
     6601 ccttcctgcg catcaatcca ctcggccagt tgccggtatt gcgcgatgat gatttcgtgt
     6661 tgcgcgatgc ccaggccatc ctggtttacc tggcgaacaa atacgacacc agtttgacct
     6721 ggtatccgac acatgcgccc gccatcctcg gccaggcaaa tatgtggctc gcgtttgccg
     6781 atggtttgac cggctcgatt tccgctgcac gtttgcatga tttgtttttt tacgatttca
     6841 atgcagagca atgccgtgcc cgcgcgcacg aactactgcg catactggac gaacacctgt
     6901 gggcggcgga acaaaatgga catcaatggc tatgtccggc cgccgcaccc accatcgcgg
     6961 atcttgcctg cttcccgtat atcgcattgt cggatgaagc cggcgtttca ctactcgact
     7021 acccggcggt acgacgctgg ctggaccgtg tcaaacgcat ccccggattc accgtcatgt
     7081 cggggatttt tccaacctca gcttccttcg atccggcctc acttgcggaa gaaacctgtc
     7141 aataagtaat gatactagag tactagtagc ggccgctgca gtccggcaaa aaagggcaag
     7201 gtgtcaccac cctgcccttt ttctttaaaa ccgaaaagat tacttcgcgt tatgcaggct
     7261 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     7321 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     7381 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccac
     7441 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     7501 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     7561 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     7621 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     7681 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     7741 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     7801 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     7861 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     7921 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     7981 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     8041 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     8101 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     8161 taaagtatat atgagtaaac ttggtctgac agctcgaggc ttggattctc accaataaaa
     8221 aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact
     8281 ggatctatca acaggagtcc aagcgagctc gatatcaaat tacgccccgc cctgccactc
     8341 atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc
     8401 atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc
     8461 catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt
     8521 gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa
     8581 ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg
     8641 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac
     8701 ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg
     8761 aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
     8821 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
     8881 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt
//
