LOCUS       sequence-193694-        6903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the Beta-1,3-1,4-Endo-Laminarinase FaGH16A in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6903)
  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 6903)
  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(+)/FaGH16A
FEATURES             Location/Qualifiers
     source          1..6903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1390..1409
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1390..1408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1409..1433
                     /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             1448..1470
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1489..1506
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1516..1533
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3157..3174
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3227..3245)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3241..3288
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3325..3780
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3412..3431)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3622..3643
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3872..4687)
                     /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(3872..4687)
                     /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     4595..4614
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4679..4698
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4809..5397
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5298..5317
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5551..5568
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5583..5722
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5648..5667)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5808..5830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5824..6015)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6824..6903,1..1003))
                     /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 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1381 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1441 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
     1501 catcacagca gcggcctggt gccgcgcggc agccatatgg ctagcgaaga cgatgtgtcc
     1561 ataggtgaaa ttatggcgcc tacggctgta acaatatcag cagaaatagt gggtgtagat
     1621 accgataatc catatggaga tggtagtgga gttgtcgttt ttacagctac tgcagataac
     1681 gaaatcactt atcaattcga ttttggagat ggaaaaacag gagttgcgcc ttcagggata
     1741 accgaacacc gatttactac aacaggtatc aatacatata ctgtagtggt taatgctgtt
     1801 ggaacaggtg gcgttaaatc gagtacggcc atggatgtag aagtgtttag ttcgttctct
     1861 gatgttgaag cagaaaattt aatggcagga gagttagttg gagattctag aacttggtat
     1921 tgggctgcaa atattccact tcatgttgga ttaggtccaa ttgaagacga ttatggaaat
     1981 ggtgagtttg cttacgaagc ttggtggaat gccattcaac catgggatga agagaaatat
     2041 tgtatgtata cgaatgaatt tgtctttaca cgtacaagtg agggattaac tttcgagcaa
     2101 acagtaggac ctgcttttgt tcctggtgct tatgcagacg aactaggtat agctggagat
     2161 acttgctatg atgaaactgt ggctacagga atgtttggtg ttaaaaatgt ttccttttta
     2221 ccatcaacgt caaaagccgc tttagaagga agttataacg atatgcctta tagaggaacc
     2281 gctttcgaaa tatctgatgg tggatttatg ggctggtacg ttggagcgag tagctacgat
     2341 attatttcag taacagaaaa tacacttcat gttagaatca ttcaacctgg atttggagcg
     2401 tggtatcaag tgttcacttc tagtaaacct gttgaagggg aagttgattt tgaatccgaa
     2461 tttaatgact tagtgtggtc cgatgagttt gatacttcag gagctccaga tgcagcaaac
     2521 tggacctacg atttaggtgc aggtggttgg ggaaaccaag aagtgcaaac ttatactaat
     2581 gatgccaata atgtaattgt agaagatgga attttaaaaa tcaaggtgct aaaagaagga
     2641 aacacttata cgtctgctag attaaaatct cagggattac aatcgtttaa atatgggcgt
     2701 atagaggcta aagcaaaatt accggcggct caaggcacgt ggccagcaat ttggatgcta
     2761 ggatctaatt ttgaaagtgt gggatggcca ttatgtggag aaatcgacat tatggaacaa
     2821 actggatggg ataaaaacac ctcactagga actttccatt ggttcgaatc gagcacaaat
     2881 gcgaatgcaa gttatggcga aactttagca gtaagtgaaa tgacatcgga attccattta
     2941 tatactttag agtggtctga agagcattta attattctag tagataatgt tcaagtggtg
     3001 aaattagaca atagtgaagc cttaccattt tacgataaga atttttttat gatacttaat
     3061 gtagccatgg gcggaagttt aggaggagat atagatcctg actttactac cgattctatg
     3121 gaaatagatt atgtaagagt ttaccaataa ctcgagcacc accaccacca ccactgagat
     3181 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     3241 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     3301 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3361 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3421 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3481 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3541 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3601 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3661 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3721 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3781 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3841 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     3901 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3961 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     4021 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     4081 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     4141 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     4201 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     4261 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     4321 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     4381 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     4441 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     4501 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     4561 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     4621 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     4681 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     4741 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4801 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4861 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4921 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4981 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5041 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5101 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5161 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5221 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5281 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5341 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5401 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5461 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5521 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5581 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     5641 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     5701 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     5761 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5821 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     5881 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5941 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     6001 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     6061 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     6121 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     6181 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     6241 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     6301 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     6361 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     6421 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     6481 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     6541 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     6601 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     6661 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     6721 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     6781 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     6841 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6901 ttt
//
