LOCUS       sequence-49428-e        4164 bp    DNA     circular SYN 15-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4164)
  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 4164)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pRAJ23m
FEATURES             Location/Qualifiers
     source          1..4164
                     /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"""
     protein_bind    54..70
                     /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             147..854
                     /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(171..199)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     794..814
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      877..948
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      964..991
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    998..1018
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(1102..1119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1273..1861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1353..1372)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2094..2909)
                     /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(2094..2909)
                     /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     2817..2836
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2901..2920
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(join(3064..3855,3856..3924))
                     /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     3687..3706
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3925..4029)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4097..4115
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4119..4162)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattcgcgg ccgcttctag agctagcata aatgtgagcg gataacattg acattgtgag
       61 cggataacaa gatactgagc acgtgtcggt tgtatttctt ctttaggcac cctcttaaag
      121 aggagatgta atgtatgact agccgtaaag gagaagaact tttcactgga gttgtcccaa
      181 ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg
      241 aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttgcactact ggaaaactac
      301 ctgttccatg gccaacactt gtcactactt tcggttatgg tgttcaatgc tttgcgagat
      361 acccagatca tatgaaacag catgactttt tcaagagtgc catgcccgaa ggttatgtac
      421 aggaaagaac tatatttttc aaagatgacg ggaactacaa gacacgtgct gaagtcaagt
      481 ttgaaggtga tacccttgtt aatagaatcg agttaaaagg tattgatttt aaagaagatg
      541 gaaacattct tggacacaaa ttggaataca actataactc acacaatgta tacatcatgg
      601 cagacaaaca aaagaatgga atcaaagtta acttcaaaat tagacacaac attgaagatg
      661 gaagcgttca actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc
      721 ttttaccaga caaccattac ctgtccacac aatctgccct ttcgaaagat cccaacgaaa
      781 agagagacca catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg
      841 atgaactata caaataataa tactagagcc aggcatcaaa taaaacgaaa ggctcagtcg
      901 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac
      961 actggctcac cttcgggtgg gcctttctgc gtttatatac tagtagcggc cgctgcagtc
     1021 cggcaaaaaa acgggcaagg tgtcaccacc ctgccctttt tctttaaaac cgaaaagatt
     1081 acttcgcgtt atgcaggctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1141 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     1201 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1261 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1321 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1381 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1441 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1501 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1561 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1621 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1681 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     1741 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1801 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1861 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1921 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1981 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gctcgagtcc
     2041 cgtcaagtca gcgtaatgct ctgccagtgt tacaaccaat taaccaattc tgattagaaa
     2101 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     2161 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     2221 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     2281 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     2341 ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta
     2401 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     2461 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     2521 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     2581 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     2641 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     2701 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     2761 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     2821 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctggag
     2881 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     2941 gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt
     3001 tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa ggatcagctc
     3061 gagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3121 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3181 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3241 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3301 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3361 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3421 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3481 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3541 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3601 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3661 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3721 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3781 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3841 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3901 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     3961 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4021 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     4081 gacattaacc tataaaaata ggcgtatcac gaggcagaat ttcagataaa aaaaatcctt
     4141 agctttcgct aaggatgatt tctg
//
