LOCUS       sequence-49420-e        5238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5238)
  AUTHORS   Rodrigo G, Landrain TE, Jaramillo A
  TITLE     De novo automated design of small RNA circuits for engineering 
            synthetic riboregulation in living cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15271-6. Epub 2012 Sep
            4.
  PUBMED    22949707
REFERENCE   2  (bases 1 to 5238)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2012-09-18"; volume: "109(38)"; pages: 
            "15271-6. Epub 2012 Sep 4"
COMMENT     Sequence Label: pRAJ31
FEATURES             Location/Qualifiers
     source          1..5238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..22
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(26..703)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             710..721
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    953..971
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    978..996
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1035..1051
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1098..1109
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1125..1835
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="RKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     misc_feature    1842..1862
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1898..1929
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(2028..2843)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2751..2770
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2835..2854
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3201..4151)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      4199..4421
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(4913..5007)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      complement(5163..5192)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
ORIGIN
        1 gaattcgcgg ccgcttctag agttattaag caccggtgga gtgacgacct tcagcacgtt
       61 cgtactgttc aacgatggtg tagtcttcgt tgtgggaggt gatgtccagt ttgatgtcgg
      121 ttttgtaagc acccggcagc tgaaccggtt ttttagccat gtaggtggtt ttaacttcag
      181 cgtcgtagtg accaccgtct ttcagtttca gacgcatttt gatttcacct ttcagagcac
      241 cgtcttccgg gtacatacgt tcggtggaag cttcccaacc catggttttt ttctgcataa
      301 ccggaccgtc ggacgggaag ttggtaccac gcagtttaac tttgtagatg aactcaccgt
      361 cttgcaggga ggagtcctgg gtaacggtaa caacaccacc gtcttcgaag ttcataacac
      421 gttcccattt gaaaccttcc gggaaggaca gtttcaggta gtccgggatg tcagccgggt
      481 gtttaacgta agctttggaa ccgtactgga actgcgggga caggatgtcc caagcgaacg
      541 gcagcggacc acctttggta actttcagtt tagcggtctg ggtaccttcg tacggacgac
      601 cttcaccttc accttcgatt tcgaactcgt gaccgttaac ggaaccttcc atacgaactt
      661 tgaaacgcat gaactctttg ataacgtctt cggaggaagc catctagtat ttctcctctt
      721 tctctagtgg atccgctagc atcgatgcga aaaaaccccg ccgaagcggg gttttttgcg
      781 gctagcaaaa aaagatcgta gccatgtagc aaatagctac taagaccaat acagtgacag
      841 cggataggga cttaattgcg gatgagacga cggtaaagct taatattgat cctagcaacc
      901 atttaccaca taactacgac ccaggcgctg gacgtttgat gtgtgctcag tatctctatc
      961 actgataggg atgtcaatct ctatcactga tagggaccta ggatgcatat aaatgtgagc
     1021 ggataacatt gacattgtga gcggataaca agatactgag cacagtggat atgctttacc
     1081 tcttggtgag acgacataaa gaggagaaac ataatatgac tagccgtaaa ggcgaagagc
     1141 tgttcactgg tgtcgtccct attctggtgg aactggatgg tgatgtcaac ggtcataagt
     1201 tttccgtgcg tggcgagggt gaaggtgacg caactaatgg taaactgacg ctgaagttca
     1261 tctgtactac tggtaaactg ccggtacctt ggccgactct ggtaacgacg ctgacttatg
     1321 gtgttcagtg ctttgctcgt tatccggacc atatgaagca gcatgacttc ttcaagtccg
     1381 ccatgccgga aggctatgtg caggaacgca cgatttcctt taaggatgac ggcacgtaca
     1441 aaacgcgtgc ggaagtgaaa tttgaaggcg ataccctggt aaaccgcatt gagctgaaag
     1501 gcattgactt taaagaagac ggcaatatcc tgggccataa gctggaatac aattttaaca
     1561 gccacaatgt ttacatcacc gccgataaac aaaaaaatgg cattaaagcg aattttaaaa
     1621 ttcgccacaa cgtggaggat ggcagcgtgc agctggctga tcactaccag caaaacactc
     1681 caatcggtga tggtcctgtt ctgctgccag acaatcacta tctgagcacg caaagcgttc
     1741 tgtctaaaga tccgaacgag aaacgcgatc atatggttct gctggagttc gtaaccgcag
     1801 cgggcatcac gcatggtatg gatgaactgt acaaatgatg atactagtag cggccgctgc
     1861 aggagtcact aagggttagt tagttagatt agcagaaagt caaaagcctc cgaccggagg
     1921 cttttgacta aaacttccct tggggttatc attggggctc actcaaaggc ggtaatcaga
     1981 taaaaaaaat ccttagcttt cgctaaggat gatttctgct agtattatta gaaaaactca
     2041 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     2101 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccaaag aatggcaagg
     2161 tcctggtaac ggtctgcgat tccgacccgt ccaacatcaa tacaacctat taatttcccc
     2221 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     2281 aatggcaaga gcttgtgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     2341 tcatcaaaat cactcgcatc aaccaaaccg ttattcatgc gtgattgcgc ctgagcaaga
     2401 cgaaatacac gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg taaccggcgc
     2461 aggaacacgg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     2521 tggaaggctg ttttcccagg aatcgcggtg gtgagtaacc acgcatcatc aggagtacgg
     2581 ataaaatgct tgatggtcgg gagaggcata aactccgtca gccagttgag acggaccatc
     2641 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     2701 tcgggcttcc catacaagcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     2761 catttatacc catataaatc agcgtccatg ttggagttta agcgcggacg ggagcaagac
     2821 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     2881 tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac
     2941 acaacgtggc tttgttgaat aaatcgaact tttgctgagt tgaaggatca gctctagtag
     3001 ttacattgtc gatctgttca tggtgaacag ctttaaatgc accaaaaact cgtaaaagct
     3061 ctgatgtatc tatctttttt acaccgtttt catctgtgca tatggacagt tttccctttg
     3121 atatctaacg gtgaacagtt gttctacttt tgtttgttag tcttgatgct tcactgatag
     3181 atacaagagc cataagaacc tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg
     3241 gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatgctt actttgcatg
     3301 tcactcaaaa attttgcctc aaaactggtg agctgaattt ttgcagttaa agcatcgtgt
     3361 agtgtttttc ttagtccgtt acgtaggtag gaatctgatg taatggttgt tggtattttg
     3421 tcaccattca tttttatctg gttgttctca agttcggtta cgagatccat ttgtctatct
     3481 agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac caccaatttc
     3541 atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg gttaagcctt
     3601 ttaaactcat ggtagttatt ttcaagcatt aacatgaact taaattcatc aaggctaatc
     3661 tctatatttg ccttgtgagt tttcttttgt gttagttctt ttaataacca ctcataaatc
     3721 ctcatagagt atttgttttc aaaagactta acatgttcca gattatattt tatgaatttt
     3781 tttaactgga aaagataagg caatatctct tcactaaaaa ctaattctaa tttttcgctt
     3841 gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa aggattcctg
     3901 atttccacag ttctcgtcat cagctctctg gttgctttag ctaatacacc ataagcattt
     3961 tccctactga tgttcatcat ctgagcgtat tggttataag tgaacgatac cgtccgttct
     4021 ttccttgtag ggttttcaat cgtggggttg agtagtgcca cacagcataa aattagcttg
     4081 gtttcatgct ccgttaagtc atagcgacta atcgctagtt catttgcttt gaaaacaact
     4141 aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca attgagatgg
     4201 gctagtcaat gataattaca tgtccttttc ctttgagttg tgggtatctg taaattctgc
     4261 tagacctttg ctggaaaact tgtaaattct gctagaccct ctgtaaattc cgctagacct
     4321 ttgtgtgttt tttttgttta tattcaagtg gttataattt atagaataaa gaaagaataa
     4381 aaaaagataa aaagaataga tcccagccct gtgtataact cactacttta gtcagttccg
     4441 cagtattaca aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag accttaaaac
     4501 cctaaaggct taagtagcac cctcgcaagc tcgggcaaat cgctgaatat tccttttgtc
     4561 tccgaccatc aggcacctga gtcgctgtct ttttcgtgac attcagttcg ctgcgctcac
     4621 ggctctggca gtgaatgggg gtaaatggca ctacaggcgc cttttatgga ttcatgcaag
     4681 gaaactaccc ataatacaag aaaagcccgt cacgggcttc tcagggcgtt ttatggcggg
     4741 tctgctatgt ggtgctatct gactttttgc tgttcagcag ttcctgccct ctgattttcc
     4801 agtctgacca cttcggatta tcccgtgaca ggtcattcag actggctaat gcacccagta
     4861 aggcagcggt atcatcaaca ggcttacccg tcttactgtc cctagtgctt ggattctcac
     4921 caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agttctgagg
     4981 tcattactgg atctatcaac aggagtccaa gcgagctcgt aaacttggtc tgacagctct
     5041 agctccggca aaaaaacggg caaggtgtca ccaccctgcc ctttttcttt aaaaccgaaa
     5101 agattacttc gcgtttgcca cctgacgtct aagaaaagga atattcagca atttgcccgt
     5161 gccgaagaaa ggcccacccg tgaaggtgag ccagtgagtt gattgctacg taattagtta
     5221 gttagccctt agtgactc
//
