LOCUS       sequence-13331-e        6364 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of photoactivated cyclase bPAC of Beggiatoa sp. optimized
            for E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6364)
  AUTHORS   Stierl M, Stumpf P, Udwari D, Gueta R, Hagedorn R, Losi A, Gartner 
            W, Petereit L, Efetova M, Schwarzel M, Oertner TG, Nagel G, Hegemann
            P
  TITLE     Light modulation of cellular cAMP by a small bacterial 
            photoactivated adenylyl cyclase, bPAC, of the soil bacterium 
            Beggiatoa.
  JOURNAL   J Biol Chem. 2011 Jan 14. 286(2):1181-8.
  PUBMED    21030594
REFERENCE   2  (bases 1 to 6364)
  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: "J Biol 
            Chem. 2011 Jan 14. 286(2):1181-8."
COMMENT     Sequence Label: pET28a-ec_bPAC
FEATURES             Location/Qualifiers
     source          1..6364
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..27
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(47..124)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     330..349
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     433..452
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        433..451
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    452..476
                     /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             491..513
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             532..549
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             559..576
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1657..1674
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1727..1745)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1741..1788
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1825..2280
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1912..1931)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2122..2143
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2373..3188)
                     /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(2373..3188)
                     /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     3096..3115
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3180..3199
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3310..3898
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3799..3818
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4052..4069
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4084..4226
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4152..4171)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4312..4334
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4328..4519)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5328..6364,1..46))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
       61 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
      121 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
      181 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
      241 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
      301 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
      361 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
      421 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      481 tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat
      541 catcatcaca gcagcggcct ggtgccgcgc ggcagccata tgatgaaacg cctggtgtat
      601 attagcaaaa ttagcggcca tctgagcctg gaagaaattc agcgtattgg caaagtgagc
      661 atcaaaaaca accagcgcga taacattacc ggcgtgctgc tgtatctgca gggcctgttt
      721 tttcagattc tggaaggcga aaacgaaaaa gtggataaac tgtataaaaa aatcctggtg
      781 gatgatcgtc ataccaacat tctgtgcctg aaaaccgaat atgatatcac cgatcgcatg
      841 tttccgaact gggccatgaa aaccattaac ctgaacgaaa acagcgaact gatgattcag
      901 ccgattaaaa gcctgctgca gaccattacc cagagccatc gtgtgctgga aaaatatatg
      961 ccggcacgtg tgatttatct gattaaccag ggcattaatc cgctgaccgt ggaaccgcag
     1021 ctggttgaaa aaatcatctt cttcagcgat attctggcct ttagcaccct gaccgaaaaa
     1081 ctgccggtga acgaagtggt gattctggtg aaccgctatt ttagcatttg cacccgtatt
     1141 attagcgcgt atggcggcga agtgaccaaa tttattggcg attgcgtgat ggcgagcttt
     1201 accaaagaac agggtgatgc ggcgattcgt accagcctgg atattatcag cgaactgaaa
     1261 cagctgcgtc atcatgtgga agcgaccaat ccgctgcatc tgctgtatac cggcattggc
     1321 ctgagctatg gccatgtgat tgaaggcaac atgggcagca gcctgaaaat ggatcacacc
     1381 ctgctgggtg atgcggtgaa cgttgcggcg cgtctggaag cgctgacccg tcagctgccg
     1441 tatgcgctgg cctttaccgc gggtgtgaaa aaatgctgcc aggcgcagtg gacctttatt
     1501 aacctgggcg cgcatcaggt gaaaggcaaa caggaagcga ttgaagttta taccgtgaac
     1561 gaagcgcaga aatattatga taccctgcag attacccagc tgattcgtca gaccctggaa
     1621 aacgataaat aataaaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
     1681 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1741 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1801 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1861 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1921 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1981 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2041 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2101 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2161 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2221 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     2281 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2341 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     2401 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     2461 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     2521 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     2581 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     2641 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     2701 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     2761 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     2821 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     2881 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     2941 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     3001 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     3061 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     3121 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     3181 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     3241 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3301 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3361 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3421 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3481 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3541 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3601 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3661 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3721 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3781 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3841 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3901 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3961 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4021 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4081 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg
     4141 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4201 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4261 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4321 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     4381 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     4441 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     4501 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     4561 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4621 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     4681 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     4741 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     4801 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     4861 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     4921 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     4981 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     5041 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     5101 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     5161 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     5221 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     5281 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     5341 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5401 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     5461 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     5521 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     5581 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     5641 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     5701 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     5761 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     5821 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     5881 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     5941 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     6001 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     6061 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     6121 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     6181 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     6241 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     6301 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     6361 taag
//
