LOCUS       sequence-174178-        6741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the Beta-1,6-Exo-Laminarinase FbGH30 in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6741)
  AUTHORS   Becker S, Scheffel A, Polz MF, Hehemann JH
  TITLE     Accurate quantification of laminarin in marine organic matter with 
            enzymes from marine microbes.
  JOURNAL   Appl Environ Microbiol. 2017 Feb 17. pii: AEM.03389-16. doi: 
            10.1128/AEM.03389-16.
  PUBMED    28213541
REFERENCE   2  (bases 1 to 6741)
  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: "Appl 
            Environ Microbiol. 2017 Feb 17. pii: AEM.03389-16. doi: 
            10.1128/AEM.03389-16."
COMMENT     Sequence Label: pET28a(+)/FbGH30
FEATURES             Location/Qualifiers
     source          1..6741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      14..602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     503..522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     756..773
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    788..927
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(853..872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1013..1035
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1029..1220)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2029..3111)
                     /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     3073..3092
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3112..3189)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3395..3414
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3498..3517
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3498..3516
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3517..3541
                     /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             3556..3578
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3597..3614
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3624..3641
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5103..5120
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5173..5191)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5187..5234
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5271..5726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5358..5377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5568..5589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5818..6633)
                     /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(5818..6633)
                     /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     6541..6560
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6625..6644
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
       61 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      121 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
      181 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      241 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      301 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      361 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      421 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      481 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      541 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      601 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      661 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      721 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
      781 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
      841 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
      901 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
      961 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     1021 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     1081 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     1141 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     1201 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     1261 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     1321 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     1381 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     1441 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     1501 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     1561 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     1621 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     1681 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     1741 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     1801 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     1861 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     1921 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     1981 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     2041 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     2101 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     2161 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     2221 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     2281 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     2341 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     2401 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     2461 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     2521 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     2581 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     2641 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     2701 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     2761 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     2821 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     2881 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     2941 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     3001 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     3061 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     3121 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     3181 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     3241 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     3301 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     3361 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     3421 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     3481 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     3541 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
     3601 atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatggct agcctcacag
     3661 catgtacatc taaagaacct ttgcaagtaa ccgtttatga aacgtctgcg aatggtaaca
     3721 agttaaccga agtatcagaa tttagtactt ctgaaaaccc ttcgaaaatt caactgagtc
     3781 cggaacaaat attccaaacc atcactggtt ttgggggctc atttacggaa tcttctgcgt
     3841 acttactaaa tcgtttgagt aaaaaaaata gaactaaaat tatcgacgct tactttagtg
     3901 aaaaaggtgc caactattca ttgacccgta cacatattag ttcttgtgat ttttcattaa
     3961 ataactatac ctatgcgcct atagcggatg attttgagct aaaacatttc agcatcatag
     4021 aagatatgga cgatttaatc cctatgataa aagacgcaat ggccgtctct gaagatggat
     4081 ttaaaatcat agcttctcca tggaccgcac cgccttggat gaaagataat aaagaatatg
     4141 ttggtggtaa attactacca gaatactacg atacctttgc cttatttttt tctaaataca
     4201 tagattatta taaagctgaa ggcattgata tttggggttt aacccccgtc aatgagcctc
     4261 atggaaatgg caataactgg gagagtatgc atttctctcc agaagaggaa accgattttg
     4321 ttcaaaacca tttaggacca aaactggaag cggacggaaa aggagcaatc aatattttag
     4381 gctatgatca aaacagagct ggattaaaag aatgggtcga tgtgatgttt gaaaatgaag
     4441 cagcttccaa gtattatgca ggaaccgcca ttcattggta tgaaagtact tatgattttt
     4501 tccctaaagc attacaatat gctcatgaca aagcacctaa caaatactta atagaaaccg
     4561 aaggctgtgt agattctgaa attccaaaat ggcaagatga caaatggtat tggtctaaag
     4621 aggcaacgga ttggggattt gattggcgag agcaagaaaa aaaatattta catccaaaat
     4681 acgcccctgt caacagatat gccagagata ttattggttg cttaaataac tgggttgacg
     4741 gttgggtcga ttggaatatg gtattggaca gacagggtgg tccaaactgg tttaaaaact
     4801 ggtgtgttgc tcctgtgatt gtagattccg ataacgatga agtctatttt acgcctttgt
     4861 attatacgat ggcacatttt agtaaataca tacgacccgg cgccaaagtg attggtctgg
     4921 atcattcgga taccgaccta caagtcaccg ctgcacaaaa ccctgatgga accattgcag
     4981 ttgtggtatt caatgaagga acaacattaa aaacctttga actttcttta ggagaatctt
     5041 caacaataat agccatacaa ccacaagcca tacaaacgat tgtattacca aactaactcg
     5101 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     5161 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     5221 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     5281 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5341 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5401 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5461 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5521 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5581 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5641 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5701 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     5761 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     5821 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     5881 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     5941 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     6001 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     6061 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     6121 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     6181 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     6241 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     6301 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     6361 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     6421 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     6481 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     6541 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     6601 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     6661 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6721 cgtcagaccc cgtagaaaag a
//
