LOCUS       sequence-424932-        6902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Designed for cytoplasmic E.coli overexpression of glucanase 
            (beta-1,3-glycosidase) for gentle yeast spheroblasting that unlike 
            Zymolyase prevents proteolysis of intracellular components. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6902)
  AUTHORS   Kuznetsov VI, Haws SA, Fox CA, Denu JM
  TITLE     General method for rapid purification of native chromatin fragments.
  JOURNAL   J Biol Chem. 2018 Aug 3;293(31):12271-12282. doi: 
            10.1074/jbc.RA118.002984. Epub 2018 May 24.
  PUBMED    29794135
REFERENCE   2  (bases 1 to 6902)
  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: 
            "10.1074/jbc.RA118.002984"; journalName: "J Biol Chem"; date: 
            "2018-08-3- 3"; volume: "293"; issue: "31"; pages: "12271-12282"
COMMENT     Sequence Label: pT22-GluCNZ-6His
FEATURES             Location/Qualifiers
     source          1..6902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1710..1732
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1747..1771
                     /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(1771..1790)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1772..1790)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1874..1893)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2099..2176
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2177..3259
                     /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(2196..2215)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4068..4259
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4253..4275)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4361..4500
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4416..4435
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4515..4532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4686..5274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4766..4785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5445..6236,6237..6305))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6068..6087
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6306..6410)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6436..6891)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6573..6594)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6785..6804
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gaggagcgtc cagcgctggg
      181 cctcggtctg gttgcaggtc cagagctgga ccttctggcc gtcacgcagc ggtgcgccgc
      241 cctgggcgtc gaggcacttg ccggactgcg ggttgcgcag ggccttggtc gcggagtcgt
      301 acgtccactt ctgcgcgccc gtcccgttgc acgtgtagat ccacacggcg gtgccgtcgg
      361 ccgtcccgct gcgcgcgacg tcgagacact tgccgagggc gcgcaccgtc ccgtcggtgc
      421 cgcgcgtcca ctgctgcgcc gcgttgccgc tgcaggtggc gagctggacc tggttggtgt
      481 cggtcgggtc ggcccacggg acgtcgaggc acagcgtgct cccgatgcgg agcgcgcccg
      541 tcccggcggg cagaccgccg ggggtgcccg gctgctcggt gctgccgccg ttcccgaggg
      601 gcgtggccgt gccggtgaac gggtcgagct tgatgtacgc ggcctggggg ttgccgtcgt
      661 ggacgagcga ctcgtgcgcg ccgacgtcgt cgaacgcgaa cgcgtacgcc ttgccgttcg
      721 ccatctgctg gtgcgcgagc ttcgcgtaga cgttggtgcg cgcgtcctgg tagaagccgg
      781 cgctgttcgc gtccggctgg ttcgggttgg tcagcgccgt ggtgcggacg agcgcggcgc
      841 acagggtgcg ggcgatcggc ccgacgacgt ggtcgttggg cgcctggagg tccttgtggc
      901 acccgtacac gctcgacgcg tcgggcttct tgaacgcggc gacctcctgc ccggacccgc
      961 tcctgaagcg gaaccagtcg ccgtcgaccc ggccgcggaa ctgcgtgccg ggctcgtggg
     1021 agaacggcgt gacgaccatg tcgcgcgtgc ggtacgagtt ccacacctcg gtgacgtagg
     1081 agtcgatcga ggccgacgag atcttcccga cgtcgatccc gtgcgacggg ttgagcgcgc
     1141 gcaggcggct cccgtcgggc gcgcgctgca cgagcccgcc ccaccccgcg ccctcgaggg
     1201 ccgtgtagaa cgcctcgtag ccgttcggct tgagcatgcc cgtgctgagg acctgcccgt
     1261 cggcgcgctg gacgcccacc tggtacggcg cggaccagtg gtccacctgc gtgctgttga
     1321 tccacaggcc gccgtcgttg agcgtgtact cggtccagtt gaagaggatg ttccggttgg
     1381 ggtcggagtc gttctggacg gcgggctgga cgagccgccc gtcgagcacg atctggaacg
     1441 tcatcttctg gccgtacgag tagtagacgc gcccggacag cttcgggagt cggatcgtca
     1501 cggactggcc ggggccgggg cccgcgatgg acgcgtcggg tgccgggacg ggcacgggcc
     1561 cgaccccgcc gggccacggg tggaacgtgc cgcccgcgtc cgcccagccg gcgacgccgt
     1621 cgcgctcgcc gaggacgtac aggtagatcg gccccctgcc cgagtcgttg gtgatcgtca
     1681 gcgggatggt cgcggggacc atatgtatat ctccttctta aagttaaaca aaattatttc
     1741 tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg
     1801 atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     1861 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
     1921 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
     1981 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
     2041 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga
     2101 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     2161 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     2221 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     2281 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     2341 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     2401 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     2461 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     2521 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     2581 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     2641 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     2701 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     2761 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     2821 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     2881 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     2941 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     3001 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     3061 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     3121 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     3181 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     3241 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     3301 cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     3361 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     3421 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
     3481 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
     3541 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc
     3601 cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
     3661 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
     3721 aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc
     3781 tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
     3841 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
     3901 tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca
     3961 gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg
     4021 gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg
     4081 aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga
     4141 aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg
     4201 ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     4261 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     4321 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     4381 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     4441 agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4501 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4561 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4621 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4681 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4741 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4801 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4861 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4921 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4981 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5041 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5101 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     5161 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5221 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5281 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5341 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5401 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5461 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5521 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5581 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5641 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5701 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5761 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5821 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5881 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5941 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6001 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6061 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6121 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6181 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6241 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6301 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6361 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6421 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     6481 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     6541 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     6601 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     6661 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     6721 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     6781 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     6841 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     6901 ca
//
