LOCUS       sequence-404714-        7764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  exo-beta mannosidase useful in enzymatic biotransformations of 
            glycans and in coupled assays of various carbohydrate processing 
            enzymes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7764)
  AUTHORS   Deng L, Tsybina P, Gregg KJ, Mosi R, Zandberg WF, Boraston AB, 
            Vocadlo DJ
  TITLE     Synthesis of 4-methylumbelliferyl 
            alpha-d-mannopyranosyl-(1-->6)-beta-d-mannopyranoside and 
            development of a coupled fluorescent assay for GH125 
            exo-alpha-1,6-mannosidases.
  JOURNAL   Bioorg Med Chem. 2013 Aug 15;21(16):4839-45. doi: 
            10.1016/j.bmc.2013.05.062. Epub 2013 Jun 11.
  PUBMED    23816041
REFERENCE   2  (bases 1 to 7764)
  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"; doi: 
            "10.1016/j.bmc.2013.05.062"; journalName: "Bioorg Med Chem"; date: 
            "2013-08-15- 15"; volume: "21"; issue: "16"; pages: "4839-45"
COMMENT     Sequence Label: pET28Man
FEATURES             Location/Qualifiers
     source          1..7764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     558..577
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(597..674)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     880..899
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     983..1002
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        983..1001
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1002..1026
                     /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             1041..1063
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3611..3628
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3681..3699)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3695..3742
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3779..4234
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3866..3885)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4076..4097
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4326..5141)
                     /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(4326..5141)
                     /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     5049..5068
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5133..5152
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5263..5851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5752..5771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6005..6022
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6037..6176
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6102..6121)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6262..6284
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6278..6469)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7278..7764,1..596))
                     /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 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
       61 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
      121 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
      181 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
      241 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
      301 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
      361 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
      421 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
      481 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
      541 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
      601 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
      661 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
      721 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
      781 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
      841 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
      901 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
      961 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     1021 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tggtcaccca
     1081 ggacctctac gacggctgga ccctcacggc cgtctccgga cccgtcccgg ccgagctggc
     1141 cggcgtgcgc gtgcgggccc gcgtccccgg gacgagccac accgcgctgc tcgacgaggg
     1201 cctgatcccc gacccgtacc tcgaccgcaa cgaggacgtc ctcgcgtgga tgcggcgcac
     1261 cgactgggcc tacgagcgcg agctcgtcct cgacccggcc gccgccgacg agcgcgtcga
     1321 cctcgtcttc ggcggcatcg acaccgtcgg gaccgtcacg ttcgacggcc acgagctcgg
     1381 ccgcaccgcc aaccagcacc ggtcctaccg gttcgacgtg cgcgccctgc tgcgccccga
     1441 cacccagcgc ctgcgcgtgg acctgcgggc cgcgatcgtg cacgccgagg cggagcgcga
     1501 gcgcctcggc caccgcccgc tggcctaccc gcagccgttc aacatggtcc gcaagatggc
     1561 gtgctcgttc ggctgggact ggggccccga cctgcagacc gcgggcctgt ggaagccggt
     1621 ccgcgtcgag cggtggcgca ccgcgcggct cgcgtcggtg cgcacgcacg tcacggtcga
     1681 ccccgacggc accggccggg tgcgcgtgct cgtggacctc gagcgctcgg gcctgcccgg
     1741 tggcgacgcg cccgtcacgc tgcgcgcccg cgtcctgtcg gccgacgtcg ccgtcaccgt
     1801 gcccggcacg gccacgtcgg ccgtcgtcga gctcgaggtc ccccgggcac ccctgtggtg
     1861 gccggtcggc cacggcccgc agccgctgtc cgacctcacc gtcacgctcg cgacgcgcga
     1921 cgacgagccg ctcgactcct ggtcccggcg catcgggttc cgcacggtcg aggtcgacac
     1981 gacgcccgac gaggacggga cgccgttcac gttccgcgtc aacgggcggc cggtcttcgt
     2041 caagggcgcc aactggatcc ccgacgacca cctgctcacc cgcatcacgc gcgagcgcct
     2101 cgcgcaccgg ctcgaccagg ccgtcgaggc gaacctcaac ctgctgcgcg tctggggcgg
     2161 cggcatctac gagtccgagg acttctacga cctgtgcgac gagcgcggcc tgctcgtctg
     2221 gcaggacttc ctcctcgcgt gcgcggccta ccccgaggag cagcccatct gggacgagct
     2281 cgaggccgag gcgcgcgaga acgtcgcccg gctcacgccg cacgcctcgc tagtactgtg
     2341 gaacggcggc aacgagaacc tctggggctt catggactgg ggctggccgc aggagctcga
     2401 gggccgcacg tggggctacc ggctggcgac cgagctgctc aagggcgtcg tcgcggagct
     2461 cgacccgacg cgcccgtacg ccgacggcag cccgtactcc cccggcttcg cgctcgacga
     2521 cgtccacccg aacgacccgg accacggcac gcaccacgag tgggaggtct ggaaccgcgt
     2581 cgactactcc gcgtaccgcg acgacgtgcc gcggttctgc tccgagttcg gcttccaggg
     2641 cccgccgacg tggtcgaccc tcacgcgtgc cgtccgcgcc gacgacggcg gcccgctgac
     2701 caaggacgac ccgacgttcc tgctgcacca gaaggccgag gacggcaacg gcaagctcga
     2761 ccgcggcctc gcgccgcacc tgggcgtgcc cgccggcttc gtcgactggc actgggcgac
     2821 gcagctcaac caggcccgcg ccgtcgcgtt cgcgatcgag cactaccggt cgtggtggcc
     2881 ccggaccgcg ggcgcgatcg tgtggcagct caacgactgc tggccggtga cgtcgtgggc
     2941 cgcgatcgac ggcgacgagc gggtcaagcc gctgtggcac gccctgcgcc gcgcctacgc
     3001 cccgcgcctg ctcaccgtgc agccgcgcga cggccgcgac gagctcgcgg tcgtcaacga
     3061 cacgggtggc ctctggcagg gcacggtccg gctctcgcgg cgcacgctcg acggtgcgac
     3121 gctcgccgag gtcgagctcg ggctggccgt cggcgcgtgg tcggtcggcc tgttcgcgct
     3181 gcccgacgag gtcgccgcgc ccgacgacgc ggccggcgag gtcctggtcg tcgacctcgg
     3241 ggacgtccgc acggtccaca cgtgggcgca ggacgtcgac ctgcgcctcg accccgaccc
     3301 ggtgtccgcg accgtgtcgc cgctgcagga cggctaccgc gtcgacgtga ccgcccggac
     3361 gttcgcccgc tcggtcacgc tgcacgtgga ccgcctcgac cccgacgcga cggtcgacga
     3421 cgccctcgtc gacgtgccgg cgggcgagac gttctcgttc cacgtccgca cctccgcacg
     3481 gttcgacgcc gccgccctca cgcgcagccc cgtgctgcgc acggcgaacg acgtggtcgt
     3541 gccgcgcggc gcgacggcac cggcgggcgc cgagcgtgat ctccagcagt cccgcgcggc
     3601 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     3661 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     3721 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     3781 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3841 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3901 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3961 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4021 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4081 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4141 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4201 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     4261 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     4321 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     4381 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     4441 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     4501 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     4561 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     4621 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     4681 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     4741 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     4801 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     4861 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     4921 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     4981 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     5041 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     5101 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     5161 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     5221 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5281 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5341 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5401 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5461 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5521 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5581 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5641 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5701 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5761 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5821 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5881 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5941 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6001 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     6061 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     6121 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     6181 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     6241 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     6301 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     6361 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     6421 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     6481 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     6541 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     6601 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     6661 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     6721 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     6781 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     6841 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     6901 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     6961 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     7021 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     7081 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     7141 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     7201 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     7261 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     7321 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     7381 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     7441 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     7501 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     7561 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     7621 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     7681 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     7741 cgagtgagat atttatgcca gcca
//
