LOCUS       sequence-49413-e        4436 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 4436)
  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 4436)
  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: pRAJ11
FEATURES             Location/Qualifiers
     source          1..4436
                     /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    244..262
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    269..287
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    326..342
                     /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             389..400
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             419..1126
                     /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(443..471)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1066..1086
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1149..1220
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1236..1263
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1270..1290
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(1374..1391)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1545..2133)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1625..1644)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2366..3181)
                     /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(2366..3181)
                     /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     3089..3108
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3173..3192
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(join(3336..4127,4128..4196))
                     /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     3959..3978
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4197..4301)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4369..4387
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4391..4434)
                     /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 gggcctcgca taaatctgtc
      181 acagcatcaa cgtttccgct gaactgtgac agactcacac aatcaaccct cccgtgctca
      241 gtatctctat cactgatagg gatgtcaatc tctatcactg atagggaatg catgctagca
      301 taaatgtgag cggataacat tgacattgtg agcggataac aagatactga gcaccctcgc
      361 ataatttcac ttcttcaatc ctcccgttaa agaggagaaa ttatgaatga ctagccgtaa
      421 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
      481 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac
      541 ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac
      601 tttcggttat ggtgttcaat gctttgcgag atacccagat catatgaaac agcatgactt
      661 tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga
      721 cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat
      781 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata
      841 caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt
      901 taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag accattatca
      961 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac
     1021 acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt
     1081 tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaataat aatactagag
     1141 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     1201 gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct
     1261 gcgtttatat actagtagcg gccgctgcag tccggcaaaa aaacgggcaa ggtgtcacca
     1321 ccctgccctt tttctttaaa accgaaaaga ttacttcgcg ttatgcaggc ttcctcgctc
     1381 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1441 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     1501 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     1561 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     1621 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     1681 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     1741 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     1801 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     1861 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     1921 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     1981 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2041 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2101 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2161 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2221 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2281 tatgagtaaa cttggtctga cagctcgagt cccgtcaagt cagcgtaatg ctctgccagt
     2341 gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca
     2401 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     2461 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     2521 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     2581 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt
     2641 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     2701 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     2761 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     2821 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     2881 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     2941 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     3001 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     3061 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     3121 catccatgtt ggaatttaat cgcggcctgg agcaagacgt ttcccgttga atatggctca
     3181 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat
     3241 ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt tgttgaataa
     3301 atcgaacttt tgctgagttg aaggatcagc tcgagttacc aatgcttaat cagtgaggca
     3361 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3421 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3481 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3541 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3601 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3661 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3721 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3781 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3841 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3901 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3961 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4021 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4081 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4141 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4201 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4261 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4321 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     4381 acgaggcaga atttcagata aaaaaaatcc ttagctttcg ctaaggatga tttctg
//
