LOCUS       sequence-49423-e        4420 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 4420)
  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 4420)
  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: pRAJ21
FEATURES             Location/Qualifiers
     source          1..4420
                     /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"""
     terminator      complement(34..61)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(77..148)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    228..246
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    253..271
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    310..326
                     /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)."
     CDS             403..1110
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
                     CTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
                     KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV
                     TAAGITHGMDELYK"
     primer_bind     complement(427..455)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1050..1070
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1133..1204
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1220..1247
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1254..1274
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(1358..1375)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1529..2117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1609..1628)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2350..3165)
                     /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"
     CDS             complement(2350..3165)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /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     3073..3092
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3157..3176
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(join(3320..4111,4112..4180))
                     /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     3943..3962
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4181..4285)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4353..4371
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4375..4418)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattcgcgg ccgcttctag agctagctat aaacgcagaa aggcccaccc gaaggtgagc
       61 cagtgtgact ctagtagaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt
      121 ctttcgactg agcctttcgt tttatttgat gcctggccta gggccctgcc tagtcctacc
      181 cttctttgag tgagaactag gatcgctctc tcctccagtg ctcagtatct ctatcactga
      241 tagggatgtc aatctctatc actgataggg aatgcatgct agcataaatg tgagcggata
      301 acattgacat tgtgagcgga taacaagata ctgagcaccc ctgcctagta tctcttcttt
      361 gcttcctcca gtaaagagga gatattggtt atgactagcc gtaaaggaga agaacttttc
      421 actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct
      481 gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc
      541 actactggaa aactacctgt tccatggcca acacttgtca ctactttcgg ttatggtgtt
      601 caatgctttg cgagataccc agatcatatg aaacagcatg actttttcaa gagtgccatg
      661 cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca
      721 cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt
      781 gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta taactcacac
      841 aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga
      901 cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt
      961 ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg
     1021 aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg
     1081 attacacatg gcatggatga actatacaaa taataatact agagccaggc atcaaataaa
     1141 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     1201 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt atatactagt
     1261 agcggccgct gcagtccggc aaaaaaacgg gcaaggtgtc accaccctgc cctttttctt
     1321 taaaaccgaa aagattactt cgcgttatgc aggcttcctc gctcactgac tcgctgcgct
     1381 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1441 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1501 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1561 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1621 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1681 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1741 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1801 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     1861 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     1921 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     1981 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2041 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2101 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2161 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2221 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2281 ctgacagctc gagtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac
     2341 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     2401 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     2461 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     2521 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     2581 agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc
     2641 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     2701 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     2761 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     2821 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     2881 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     2941 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     3001 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     3061 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     3121 taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     3181 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     3241 gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa cttttgctga
     3301 gttgaaggat cagctcgagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3361 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3421 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3481 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3541 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3601 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3661 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3721 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3781 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3841 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3901 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3961 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4021 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4081 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4141 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4201 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4261 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     4321 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg cagaatttca
     4381 gataaaaaaa atccttagct ttcgctaagg atgatttctg
//
