LOCUS       sequence-310562-        7477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  see pDUAL_T5: exchanged origin of replication (pBR322) and 
            introduced bom site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7477)
  AUTHORS   Schwaiger KN, Voit A, Dobiasova H, Luley C, Wiltschi B, Nidetzky B
  TITLE     Plasmid Design for Tunable Two-Enzyme Co-Expression Promotes 
            Whole-Cell Production of Cellobiose.
  JOURNAL   Biotechnol J. 2020 Jul 15:e2000063. doi: 10.1002/biot.202000063.
  PUBMED    32668097
REFERENCE   2  (bases 1 to 7477)
  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: "Biotechnol 
            J. 2020 Jul 15:e2000063. doi: 10.1002/biot.202000063."
COMMENT     Sequence Label: pDUAL_2_T5
FEATURES             Location/Qualifiers
     source          1..7477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(9..23,24..29,30..46,47..53)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    29..45
                     /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)."
     protein_bind    61..77
                     /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             93..104
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             120..143
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     terminator      1671..1765
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        1772..1849
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             1850..2932
                     /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"
     primer_bind     complement(1869..1888)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     RBS             2942..2953
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(2960..3028,3029..3820)
                     /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     complement(3178..3197)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      complement(3863..3910)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(6392..6409)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6428..6450
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6465..6489
                     /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)."
     primer_bind     complement(6489..6508)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6490..6508)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      6545..7133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7034..7053
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7287..7304
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7319..7459
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7385..7404)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gatccaaatc ataaaaaatt tatttgcttt gtgagcggat aacaattata atagattcaa
       61 ttgtgagcgg ataacaattt cacacagaat ctaaagagga gaaaacgtcc atggcaagct
      121 ggagccaccc gcagttcgaa aagggtacca tgaaaaataa agtccaactg attacctatg
      181 cggatcgtct gggtgatggc accattaaaa gcatgacgga catcctgcgt acccgctttg
      241 atggcgttta tgacggtgtc cacattctgc cgtttttcac cccgtttgat ggcgccgacg
      301 caggtttcga tccgatcgac cataccaaag ttgatgaacg tctgggtagc tgggatgacg
      361 tcgccgaact gtctaaaacc cacaacatta tggtggatgc aatcgttaat cacatgtcat
      421 gggaatcgaa acagttccaa gatgttctgg ccaaaggcga agaatcagaa tattacccga
      481 tgtttctgac catgagcagc gtgttcccga acggtgccac ggaagaagac ctggcaggca
      541 tttatcgtcc gcgtccgggt ctgccgttta cccactacaa attcgcaggc aaaacgcgtc
      601 tggtctgggt gtcttttacc ccgcagcaag tggacatcga tacggacagt gataaaggtt
      661 gggaatatct gatgtccatt ttcgaccaaa tggcggccag ccatgtgtct tatatccgcc
      721 tggatgcggt tggctacggt gccaaagaag caggcaccag ttgctttatg accccgaaaa
      781 cgttcaaact gatttcccgt ctgcgcgaag aaggcgtgaa acgcggtctg gaaattctga
      841 tcgaagtcca cagctattac aaaaaacagg tggaaatcgc gtctaaagtg gatcgcgttt
      901 atgactttgc cctgccgccg ctgctgctgc atgccctgag taccggccac gtcgaaccgg
      961 tggcccattg gacggatatt cgtccgaaca atgctgttac cgtcctggat acgcatgacg
     1021 gcatcggcgt tattgatatc ggttcagatc agctggaccg ctcgctgaaa ggtctggtgc
     1081 cggacgaaga tgtggataac ctggtgaata cgattcatgc gaacacccac ggcgaatcac
     1141 aggcagctac cggtgcggcg gcatcgaacc tggatctgta ccaagttaat agtacctact
     1201 actccgctct gggctgtaac gatcagcact acatcgctgc gcgtgcggtg cagtttttcc
     1261 tgccgggtgt tccgcaagtc tattacgtgg gcgccctggc aggtaaaaat gatatggaac
     1321 tgctgcgcaa aaccaacaat ggtcgtgaca ttaaccgcca ttattacagc acggccgaaa
     1381 tcgatgaaaa tctgaaacgt ccggtggtta aagcactgaa cgctctggcg aaatttcgca
     1441 atgaactgga tgcttttgac ggcaccttct catataccac ggatgacgat acgagtattt
     1501 cctttacctg gcgtggtgaa acgtcgcagg ccaccctgac gttcgaaccg aaacgcggcc
     1561 tgggtgttga taataccacg ccggtcgcaa tgctggaatg ggaagacagt gctggtgacc
     1621 atcgctccga cgacctgatc gctaatccgc cggttgttgc gtaaaagctt gactcctgtt
     1681 gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg
     1741 ccgggcgttt tttattggtg agaatgaatt cgacaccatc gaatggtgca aaacctttcg
     1801 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     1861 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     1921 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     1981 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     2041 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     2101 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     2161 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     2221 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     2281 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     2341 ggacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     2401 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     2461 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     2521 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     2581 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     2641 tggtgcggat atctcggtag tgggatacga cgataccgag gacagctcat gttatatccc
     2701 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     2761 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     2821 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     2881 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gaagatctat
     2941 taaagaggag aaattaagca tgagtattca acatttccgt gtcgccctta ttcccttttt
     3001 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3061 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3121 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3181 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3241 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3301 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3361 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3421 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3481 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3541 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3601 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3661 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3721 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3781 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgcagatcc ttagcgaaag
     3841 ctaaggattt tttttactcg agcaaaaaac ccctcaagac ccgtttagag gccccaaggg
     3901 gttatgctag tgtacactac aatgtcacat cgacacgaac tgttgaccct gcgggggcat
     3961 aaggaaccaa gttgccttct acaggtgttc catcaacgac aaggcgaccg cgtgatccgt
     4021 cggtcccact gttagtgact gttatctcgt aagtggctcc acgggcgaca cgcgtaaccg
     4081 taaagctagg tacgtcaggt ccgatctgcg ggtctaccac caagccgtcg tactctgggc
     4141 gaaccccaag aaggtactgt gacacggtca caaagttcca agccgcagtc ccggtaagcc
     4201 agctgttctt tgcttcaccg tgacggaccg cctccttccc tgctatcatt tgtgcataga
     4261 catagggttc aagacggtga acgtcgctta tgtcctcgcg atacgcggga gtaattctct
     4321 tgtagtaatc aaatgcccgt ccgcctcttc cgacgacagt ttccgctatg ataacccaag
     4381 gattattgtg gcaaaatata cctccattct ctttataccc gggtggataa gtggatactt
     4441 cacccatgtg tacttggtat gtagtgtatg ctgggtattg aaggaccata ccgtgatcgg
     4501 ttgccaacat ttcgttcaca gaatcaagcg ccttaatagc gggagcgtcc gtatcttgag
     4561 gcccttcgcc tacaccgact ccagccatca ccgcgaaccc ttgcggttcg atccaaatct
     4621 tcccttcgtc atgtgcgtcg gtgccgatag ggtttccata atagtcataa gctctcaaga
     4681 accagctgcc gtcccacccg tcggtcagaa gtgcgtctct catttcagcc acatgccctc
     4741 ttgctctatc ggctacgtcg gccaacccgc gccgcgctgc caactcggca tactgctccc
     4801 catataaaac gaattgcgcg gctatgaacg ttgactccgc tacgccaccg gcttggttct
     4861 ccgttgtctg aaatgactcg cccggggttg tactgaagca attaagattc aaacaatcgt
     4921 tccaatctgc gcggccaata agaggcaaac cgtggggacc ccgatgggtt acggtaaact
     4981 caaaactacg ggtcaggtgc tcaaataacg gaacttctga ccccggttcg ttatcaaaag
     5041 gtacgggctc atctaatatg gagaagtcac cagtttcttt gatgtaggcg gcagttcccg
     5101 cgattaacca aagaggatcg tcgttaaacc cagagccaat gtcgttgtta ccgcgcttcg
     5161 ttaaaggttg gtattggtga taagcggaac cgtccgcgaa ctgggttgac gctatgtcaa
     5221 tgatgcgctc acgcgcccgc tctgggatta agtgaacaaa gcccagaagg tcttggttgc
     5281 tatcacgaaa ccccatccct ctcccaatac cggtctcgaa aaagctcgcc gagcgactca
     5341 tgttaaaggt taccatgcat tggtactgat tccatatatt aaccatccgg tccaattttt
     5401 cgtcattgga actaacggaa tacgtcgata acaaatccgt ccaataatcc ttcaaagcgg
     5461 cgaaagctgc gtcagtctgt tcggacgtag caaatcttga taataaagcg tgagctcttt
     5521 ctttattcac gacctgtttt gcgtcgtctg cccacttctc ctcatcagga ttttccacgt
     5581 acccaagcac ataaaccaat tctctggatt ctccaggcgc taaagacact gccacgctat
     5641 gagagccaat aggataccat ccacttgcaa cgctatttgc actttctccc ttaaggggaa
     5701 ctgcggcttc accaagcgag ttataggctc ccacaaatgt gtcgcgatct gtatcgaaac
     5761 cctcggcctg cgtattaacg gcgaataccg cgtagtgatc gcgtctctcc cggtactctg
     5821 tacggtgata aattgcactg ccatgcggag attcttgttc cacttcaacc tctcctatgc
     5881 tcagattacg ctggtagtta gtttgatcgt cttgcgcgtt ccaaagacaa aattcgacaa
     5941 aagaaaacaa cgtcagggat ttatacgagt cagaggtatt agtgaccgta actttctgga
     6001 cctccgcgtt ctcaccgact gggacgaaaa acaaggtctc aacccgtacc ccatttcttt
     6061 cacctgtaat ggtactgtag ccaagaccat gtctggcttc aaaatgatct aagtctgctt
     6121 taacaggaag ccacgatggc gtccaaacgt ccccaccgtc gtttacataa agataccggc
     6181 ctcccgcgtc tgctggtatg ttgttgtatc tgtatcttgt caatctacgc atcttggcat
     6241 cgcgataaaa cgaatagccg ccagcctggt gcgacaaaag actgaagaac tgttccgaac
     6301 ccagatagtt gatccaaggg tacggagtat gcggtgtggt gataacgtac tcgcgtgctt
     6361 catcgtcaaa atgaccatag cgggtcgacc cgtgatggtg atggtgatgg ctgctgccca
     6421 tatgatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct
     6481 cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgactagtag atcaaaggat
     6541 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6601 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6661 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     6721 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6781 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6841 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6901 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6961 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7021 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7081 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7141 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7201 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7261 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     7321 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     7381 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     7441 tgactgggtc atggctgcgc cccgacaccc gccaacg
//
