LOCUS       sequence-173614-        6504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the Beta-1,3-Endo-Laminarinase FaGH17A in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6504)
  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 6504)
  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(+)/FaGH17A
FEATURES             Location/Qualifiers
     source          1..6504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      29..617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     518..537
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     771..788
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    803..942
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(868..887)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1028..1050
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1044..1235)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2044..3126)
                     /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     3088..3107
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3127..3204)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3410..3429
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3513..3532
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3513..3531
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3532..3556
                     /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             3571..3593
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3612..3629
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3639..3656
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4881..4898
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4951..4969)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4965..5012
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5049..5504
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5136..5155)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5346..5367
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5596..6411)
                     /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(5596..6411)
                     /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     6319..6338
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6403..6422
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
       61 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
      121 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
      181 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
      241 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
      301 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
      361 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
      421 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
      481 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
      541 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
      601 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
      661 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
      721 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
      781 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
      841 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
      901 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
      961 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     1021 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     1081 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     1141 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     1201 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     1261 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     1321 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     1381 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     1441 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     1501 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     1561 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     1621 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     1681 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     1741 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     1801 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     1861 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     1921 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     1981 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     2041 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2101 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     2161 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     2221 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     2281 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     2341 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     2401 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     2461 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     2521 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     2581 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     2641 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     2701 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     2761 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     2821 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     2881 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     2941 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     3001 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     3061 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     3121 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     3181 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     3241 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     3301 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     3361 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     3421 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     3481 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     3541 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
     3601 tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata
     3661 tggctagctt aggatgtaac aataaaactc agattgcgca ggaattatcg gcagcagcta
     3721 ttttagggaa tccaaaatac ctggctattt cttatggcgg ttaccgtgaa accactcgcg
     3781 aggtgcaacc tactatggca gaattgaaag atgatttgaa gattctttcg gctatgggca
     3841 taaaagtttt acggacctat aatgtgcatt tagatcaggc ggctaatctg ttaaaagcga
     3901 taagtgaatt aaaagcaacg gatccaagtt ttgaaatgta cgttatgctt ggggcgtgga
     3961 tagattgtaa aaatgcattt aattctgcta acggcgagcc agaccatagt ctagaaagtg
     4021 aacgaaatgc tgttgaaata gagacggctg ttcagttaac aaaagcatat ccagacatcg
     4081 ttaaaattat tgctgtgggt aatgaagcca tggtaaaatg ggcggcgagt tattatgtac
     4141 agccgggcgt gattttaaaa tgggtacatc atttacaaga cttaaaacaa tcaggagaat
     4201 taccaactga tttatgggtt acaagctcag acaattttgc gtcttggggt ggaggcgatg
     4261 ccgaatatca tacagaagat ttaaatcaac taattaaaac tgtcgattat atctctatgc
     4321 atacctaccc aatgcacgat acccattatc attctgtgtt ttggggtgtt gaagacagtg
     4381 aagcagattt atcagacgaa gaaaagattg aagtggctat ggcacgcgct aaagcctatg
     4441 ccgtatcgca atacgatagt gtttacaatt acatgacgag tttaggcgta aataaaccaa
     4501 ttcatattgg agaaacaggt tgggcgagcg tgtctaacgc attgtatgga gaaatgggtt
     4561 cgaaggctac agatgaatat aaagaagcct tgtattacaa atctatgcga gactggacaa
     4621 acgaaaatgg aatgtcttgc ttttattttg aagctttcga cgagcagtgg aaagatgcca
     4681 ataatccctt aggttccgaa aatcattttg gccttatcaa tttaaatgga gaagccaaat
     4741 atgctttatg ggatttagtc gatcaaggcg tttttaaagg attgaccaga aatggaaagc
     4801 cgattacaaa aacttttgga ggtaataaag cggctttaat ggaaaccgtt gcagtgccac
     4861 caattaaaaa ttaactcgag caccaccacc accaccactg agatccggct gctaacaaag
     4921 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     4981 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     5041 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5101 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5161 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5221 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5281 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5341 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5401 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5461 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5521 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5581 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     5641 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     5701 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     5761 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     5821 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     5881 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     5941 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     6001 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     6061 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     6121 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     6181 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     6241 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     6301 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     6361 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     6421 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     6481 agttttcgtt ccactgagcg tcag
//
