LOCUS       sequence-49427-e        4452 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 4452)
  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 4452)
  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: pRAJ23
FEATURES             Location/Qualifiers
     source          1..4452
                     /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    260..278
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    285..303
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    342..358
                     /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             435..1142
                     /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(459..487)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1082..1102
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1165..1236
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1252..1279
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1286..1306
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(1390..1407)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1561..2149)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1641..1660)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2382..3197)
                     /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(2382..3197)
                     /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     3105..3124
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3189..3208
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(join(3352..4143,4144..4212))
                     /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     3975..3994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4213..4317)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4385..4403
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4407..4450)
                     /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 ggatatcgat catatccctt
      181 atgaccgatc tgctccacac atcctaggta tgaacgcggg agcaaacaaa tcgctttagg
      241 cgaccctccg tgctcagtat ctctatcact gatagggatg tcaatctcta tcactgatag
      301 ggaatgcatg ctagcataaa tgtgagcgga taacattgac attgtgagcg gataacaaga
      361 tactgagcac gtgtcggttg tatttcttct ttaggcaccc tcttaaagag gagatgtaat
      421 gtatgactag ccgtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat
      481 tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa
      541 catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct gttccatggc
      601 caacacttgt cactactttc ggttatggtg ttcaatgctt tgcgagatac ccagatcata
      661 tgaaacagca tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta
      721 tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata
      781 cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg
      841 gacacaaatt ggaatacaac tataactcac acaatgtata catcatggca gacaaacaaa
      901 agaatggaat caaagttaac ttcaaaatta gacacaacat tgaagatgga agcgttcaac
      961 tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca
     1021 accattacct gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca
     1081 tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat gaactataca
     1141 aataataata ctagagccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     1201 tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct
     1261 tcgggtgggc ctttctgcgt ttatatacta gtagcggccg ctgcagtccg gcaaaaaaac
     1321 gggcaaggtg tcaccaccct gccctttttc tttaaaaccg aaaagattac ttcgcgttat
     1381 gcaggcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     1441 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1501 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1561 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1621 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1681 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1741 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1801 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1861 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1921 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1981 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     2041 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2101 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2161 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2221 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2281 atcaatctaa agtatatatg agtaaacttg gtctgacagc tcgagtcccg tcaagtcagc
     2341 gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc
     2401 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2461 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2521 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2581 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2641 aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2701 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2761 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2821 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2881 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2941 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     3001 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     3061 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     3121 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctggagca agacgtttcc
     3181 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     3241 gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg
     3301 tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagctcga gttaccaatg
     3361 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3421 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3481 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3541 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3601 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3661 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3721 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3781 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3841 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     3901 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     3961 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4021 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4081 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4141 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4201 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4261 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4321 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     4381 taaaaatagg cgtatcacga ggcagaattt cagataaaaa aaatccttag ctttcgctaa
     4441 ggatgatttc tg
//
