LOCUS       sequence-49425-e        4444 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 4444)
  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 4444)
  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: pRAJ22
FEATURES             Location/Qualifiers
     source          1..4444
                     /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    252..270
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    277..295
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    334..350
                     /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             427..1134
                     /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(451..479)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1074..1094
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1157..1228
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1244..1271
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1278..1298
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(1382..1399)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1553..2141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1633..1652)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2374..3189)
                     /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(2374..3189)
                     /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     3097..3116
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3181..3200
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(join(3344..4135,4136..4204))
                     /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     3967..3986
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4205..4309)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4377..4395
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4399..4442)
                     /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 ggcttagtac acctgagtat
      181 aaactactat catactcaag catcagccgt caactagcaa tctttggccc gctaacacgt
      241 cgtgctcagt atctctatca ctgataggga tgtcaatctc tatcactgat agggaatgca
      301 tgctagcata aatgtgagcg gataacattg acattgtgag cggataacaa gatactgagc
      361 acgccgtcaa cttgtttctt ctttggccca cacgttaaag aggagataag tttaatgact
      421 agccgtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
      481 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga
      541 aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt
      601 gtcactactt tcggttatgg tgttcaatgc tttgcgagat acccagatca tatgaaacag
      661 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc
      721 aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt
      781 aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa
      841 ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga
      901 atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac
      961 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac
     1021 ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt
     1081 cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaataataa
     1141 tactagagcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1201 tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg
     1261 gcctttctgc gtttatatac tagtagcggc cgctgcagtc cggcaaaaaa acgggcaagg
     1321 tgtcaccacc ctgccctttt tctttaaaac cgaaaagatt acttcgcgtt atgcaggctt
     1381 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1441 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1501 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1561 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1621 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1681 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1741 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1801 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1861 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1921 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1981 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2041 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2101 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2161 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2221 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2281 aaagtatata tgagtaaact tggtctgaca gctcgagtcc cgtcaagtca gcgtaatgct
     2341 ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg
     2401 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     2461 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     2521 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     2581 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt
     2641 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     2701 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     2761 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     2821 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     2881 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     2941 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     3001 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     3061 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     3121 taaatcagca tccatgttgg aatttaatcg cggcctggag caagacgttt cccgttgaat
     3181 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     3241 tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg
     3301 ttgaataaat cgaacttttg ctgagttgaa ggatcagctc gagttaccaa tgcttaatca
     3361 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3421 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3481 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3541 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3601 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3661 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3721 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3781 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3841 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3901 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3961 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4021 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4081 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4141 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4201 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4261 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4321 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     4381 ggcgtatcac gaggcagaat ttcagataaa aaaaatcctt agctttcgct aaggatgatt
     4441 tctg
//
