LOCUS       sequence-394144-        5772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate assembly of expression constructs containing pBAD 
            promoter (spectinomycin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5772)
  AUTHORS   Lesley Milnes,  Mark Youles,  Josephine H. R. Maidment
  TITLE     pOPARA: Vectors for Golden Gate assembly of expression constructs 
            containing the araBAD promoter
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 5772)
  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: "bioRxiv"
COMMENT     Sequence Label: pOPARA2
FEATURES             Location/Qualifiers
     source          1..5772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(103..981)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1008..1292
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1215..1234
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    1442..1463
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1478..1483,1484..1501,1502..1508)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1516..1532
                     /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             1550..1561
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1568..2245
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2290..2361
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2377..2404
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2484..2501)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2654..2740
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2832..2859
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2878..2969
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3191..3982
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      4024..4479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4111..4130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4590..5178
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5079..5098
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5332..5349
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5515..5537
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5676..5695
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcttatcatc gatgcataat gtgcctgtca aatggacgaa gcagggattc tgcaaaccct
       61 atgctactcc gtcaagccgt caattgtctg attcgttacc aattatgaca acttgacggc
      121 tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg ccccggtgca
      181 ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc gaccgacggt
      241 ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac gttggtcctc
      301 gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca gacgcgacgg
      361 cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg ccaggtgatc
      421 gctgatgtac tgacaagcct cgcgtacccg attatccatc ggtggatgga gcgactcgtt
      481 aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga tttatcgcca gcagctccga
      541 atagcgccct tccccttgcc cggcgttaat gatttgccca aacaggtcgc tgaaatgcgg
      601 ctggtgcgct tcatccgggc gaaagaaccc cgtattggca aatattgacg gccagttaag
      661 ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc attcgcgagc
      721 ctccggatga cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa tatcacccgg
      781 tcggcaaaca aattctcgtc cctgattttt caccaccccc tgaccgcgaa tggtgagatt
      841 gagaatataa cctttcattc ccagcggtcg gtcgataaaa aaatcgagat aaccgttggc
      901 ctcaatcggc gttaaacccg ccaccagatg ggcattaaac gagtatcccg gcagcagggg
      961 atcattttgc gcttcagcca tacttttcat actcccgcca ttcagagaag aaaccaattg
     1021 tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct cgctaaccaa
     1081 accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag ccatgacaaa
     1141 aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt atttgcacgg
     1201 cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc ctacctgacg
     1261 ctttttatcg caactctcta ctgtttctcc atacccgttt ttttgggcta gcaggaggaa
     1321 ttcacaatgt gagaccgata tcaatacgca aaccgcctct ccccgcgcgt tggccgattc
     1381 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     1441 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     1501 gtatgttgtg tggaattgtg agcggataac aatttcacac atactagaga aagaggagaa
     1561 atactagatg gcttcctccg aggacgttat caaagagttc atgcgtttca aagttcgtat
     1621 ggaaggttcc gttaacggtc acgagttcga aatcgaaggt gaaggtgaag gtcgtccgta
     1681 cgaaggtacc cagaccgcta aactgaaagt taccaaaggt ggtccgctgc cgttcgcttg
     1741 ggacatcctg tccccgcagt tccagtacgg ttccaaagct tacgttaaac acccggctga
     1801 catcccggac tacctgaaac tgtccttccc ggaaggtttc aaatgggaac gtgttatgaa
     1861 cttcgaggac ggtggtgttg ttaccgttac ccaggactcc tccctgcaag acggtgagtt
     1921 catctacaaa gttaaactgc gtggtaccaa cttcccgtcc gacggtccgg ttatgcagaa
     1981 aaaaaccatg ggttgggaag cttccaccga acgtatgtac ccggaggacg gtgctctgaa
     2041 aggtgaaatc aaaatgcgtc tgaaactgaa agacggtggt cactacgacg ctgaagttaa
     2101 aaccacctac atggctaaaa aaccggttca gctgccgggt gcttacaaaa ccgacatcaa
     2161 actggacatc acctcccaca acgaggacta caccatcgtt gaacagtacg aacgtgctga
     2221 aggtcgtcac tccaccggtg cttaataacg ctgatagtgc tagtgtagat cgctactaga
     2281 gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     2341 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc
     2401 tgcgtttata ggtctcagct ttctagagtc gacctgcagg catgcaagct tggctgtttt
     2461 ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg
     2521 ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac
     2581 tcagaagtga aacgccgtag cgccgatggt agtgtggggt ttccccatgc gagagtaggg
     2641 aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     2701 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
     2761 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca
     2821 tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt
     2881 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2941 tgcttcaata atattgaaaa aggaagagta tgcgctcacg caactggtcc agaaccttga
     3001 ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt
     3061 ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg
     3121 atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa
     3181 gttaaacatc atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt
     3241 tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc
     3301 agtggatggc ggcctgaagc cacacagcga tattgatttg ctggttacgg tgaccgtaag
     3361 gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc
     3421 tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat
     3481 tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat
     3541 tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa
     3601 agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt
     3661 tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc
     3721 cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc
     3781 agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc
     3841 ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga
     3901 tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cattacgtaa aaggcgagat
     3961 caccaaggta gtcggcaaat aactgtcaga ccaagtttac tcatatatac tttagattga
     4021 tttacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4081 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4141 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4201 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     4261 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4321 gtggactctt gttccaaact tgaacaacac tcaaccctat ctcgggctat tcttttgatt
     4381 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4441 ttaacgcgaa ttttaacaaa atattaacgt ttacaattta aaaggatcta ggtgaagatc
     4501 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4561 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4621 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4681 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     4741 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4801 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4861 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4921 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4981 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5041 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5101 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5161 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5221 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5281 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     5341 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5401 atttcacacc gcatagggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     5461 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gactccggga
     5521 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag caaggagatg
     5581 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     5641 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     5701 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatctgc
     5761 tcatgtttga ca
//
