LOCUS       sequence-446624-        7706 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of His6-SUMO-Ub-mEos3.2-intein-CBD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7706)
  AUTHORS   Williams C, Dong KC, Arkinson C, Martin A
  TITLE     The Ufd1 cofactor determines the linkage specificity of 
            polyubiquitin chain engagement by the AAA+ ATPase Cdc48.
  JOURNAL   Mol Cell. 2023 Mar 2;83(5):759-769.e7. doi: 
            10.1016/j.molcel.2023.01.016. Epub 2023 Feb 2.
  PUBMED    36736315
REFERENCE   2  (bases 1 to 7706)
  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.molcel.2023.01.016"; journalName: "Mol Cell"; date: 
            "2023-03-2- 2"; volume: "83"; issue: "5"; pages: "759-769.e7"
COMMENT     Sequence Label: pAM292
FEATURES             Location/Qualifiers
     source          1..7706
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(671..898)
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             complement(1214..1231)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1241..1258)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1277..1299
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1314..1338
                     /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(1338..1357)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1339..1357)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1441..1460)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1666..1743
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1744..2826
                     /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(1763..1782)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4138..4329
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4323..4345)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4431..4570
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4486..4505
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4585..4602)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4756..5344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4836..4855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5515..6306,6307..6375))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6138..6157
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6376..6480)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6548..6566
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(6593..6598,6599..6615,6616..6621)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(6796..6843)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6839..6857
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6892..7047)
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             complement(7078..7671)
                     /codon_start=1
                     /gene="Mycobacterium xenopi gyrA"
                     /product="modified GyrA intein (Southworth et al., 1999)"
                     /label=Mxe GyrA intein
                     /note="N-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             complement(join(7675..7706,1..646))
                     /codon_start=1
                     /product="improved monomeric variant of green-to-red 
                     photoswitchable fluorescent protein EosFP (Zhang et al., 
                     2012)"
                     /label=mEos3.2
                     /translation="MSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVK
                     EGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNAR
                     NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLL
                     EGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPD
                     NARR"
ORIGIN
        1 caacagcatg ctcatacagc ttaaccttgt tgtaatcttt gtcatggctt aaaatctcaa
       61 tgcagtggtc cacaaagtgg gcgcctggta acttgacacc cttctcctta gctttgtaag
      121 tagttctgaa gtcacatcgg taatgggcat ttccttcaag caacaaagcc atctcaatat
      181 cacccgtcag cactccatca cgcacataca ttttctcagt ggagggctcc catttcagcg
      241 tcttcttctg cataactgga ccattggcgg gaaagttggt accataaaat cgaactttat
      301 tatagaaagt gtccccttcc attgttatgt cgtttctggc gttgcaaatg cccccgtctt
      361 cgaaagtcaa gcttcgttcc cacgaatacc ccttaggaaa cgactgctta aaatagtctt
      421 gtatgttgtc tggatatttg gcgaataccc tgttgccgta atggaatgca gtggtcagga
      481 tatcaaaggc aaaaggcaga ggtccgccct ctttgacttc aagatccata ctctgttttc
      541 cctcaaaagg cttgcctgta ccatctccgt cgatcacaaa gtggtgcccg tttacgttgc
      601 cttccatacg gagtttgatc ttcatgtctg gcttaatcgc actcatacca gatccactac
      661 cagagccgct gccgccacgc agacgcagaa ccaggtgcag ggtgctctcc ttttgaatgt
      721 tgtaatcgct cagggtacgg ccgtcttcca gctgtttacc cgcgaagatc agacgttgct
      781 gatccggcgg aatgccttcc ttgtcttgga ttttgctctt aacgttatcg atggtgtcgc
      841 tgctctccac ttccagggta atggttttac cggtcagggt cttaacaaag atctgcatca
      901 cggtgctgtg gccaccggtt tgctcctgat aaacttcaat cacatcctct tcctccatac
      961 ccagctcctt cggggtgtgg ttgtccgcga tacgctggcc ttcaaacagg aaacgcaggc
     1021 tgttcatcgg aacaccttga cgctggcagt agctctcttt cagtttcttc aggtgggtgg
     1081 tcattttcac cttgaagtgg atttcgctgc tgtcttgacc aataactttc agcttgatgt
     1141 attcgccttc cttcttgtcg cccaggtctt cggtgctcgg tttcgcttct tgatcgctca
     1201 tctcgagcat atggctgccg cgcggcacca ggccgctgct gtgatgatga tgatgatggc
     1261 tgctgcccat ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg
     1321 ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga
     1381 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     1441 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     1501 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     1561 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     1621 gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg
     1681 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     1741 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     1801 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     1861 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     1921 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     1981 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     2041 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     2101 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     2161 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     2221 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     2281 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     2341 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     2401 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     2461 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     2521 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     2581 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     2641 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     2701 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     2761 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     2821 cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc
     2881 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     2941 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     3001 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     3061 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     3121 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta
     3181 cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc
     3241 ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga
     3301 ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcattgg
     3361 accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg
     3421 gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag
     3481 ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca
     3541 agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag
     3601 aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc gggcagcgtt
     3661 gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     3721 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     3781 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     3841 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     3901 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     3961 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     4021 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     4081 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     4141 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     4201 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     4261 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     4321 aacctctgac acatgcagct cccggagacg gccacagctt gtctgtaagc ggatgccggg
     4381 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     4441 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     4501 ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     4561 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4621 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4681 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4741 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4801 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4861 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4921 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4981 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5041 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5101 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5161 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     5221 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5281 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5341 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5401 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5461 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5521 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5581 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5641 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5701 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5761 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5821 tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5881 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5941 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6001 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6061 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6121 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6181 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6241 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6301 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6361 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6421 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6481 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     6541 ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattctcat gtttgacagc
     6601 ttatcatcga taagctttaa tgcggtagtt tatcacagtt aaattgctaa cgcagtcagg
     6661 caccgtgtat gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc accctggatg
     6721 ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat atccggatat
     6781 agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc
     6841 tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctc attgaagctg
     6901 ccacaaggca ggaacgttgg atggttccca tcctgccaag gaggtgtggg gctgcaaaca
     6961 tttatacgtc ttgccgttat atgtgaccaa ttgtcccgca gtataagctg tgttgacctg
     7021 ccaagcggat acaccaggat ttgtcgtgag gcctgagttc agaccggtga ggccagtagc
     7081 gtggctgacg aacccgttcg tgataaacgc gtggtctgcc gtgtcgacac gaaggctata
     7141 caccggctgc acgccggcgt cggtgacact ggcgactttc gcgtagtaga accgcccgtc
     7201 ggtcagctcg tcggcgatag cttgggcgtc cgggtctcgg tggtgtgctt ccaagaaacg
     7261 caccagtcca gggacgccga ctgtgtaggt tgtgggcgca aattcgggtt tcccgcgggc
     7321 aaaacctgca cagtcgacgc tgaatgcgct gcgttgaatc accgcgtaat cgcccggctt
     7381 gatttcgtcg atcagcttcc acagcagggt cggcaccccg gcgacgtcga ccaaacacaa
     7441 caacgggtgg ttcgcggtgc ccgtcacacg cagaccttcg accgtacgca ccgtgtacac
     7501 cggatgctcg ccggagtgga acagccggtc ggcgagcacg ggattgccat gccggtcaag
     7561 gactttcagg tcgatggcgt tgtcactgtt gggccgcgca cccggcacga tgtcggcgat
     7621 gcgtaccgac tcgccctcgg gtagggcaac tagtgcatct cccgtgatgc aatatcgtct
     7681 ggcattgtca ggcaatccag aatgag
//
