LOCUS       sequence-488947-        6045 bp    DNA     circular SYN 09-OCT-2025
DEFINITION  Recombinant protein expression of PCMT1 H58 A214.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6045)
  AUTHORS   Pang EZ, Zhao B, Flowers C, Oroudjeva E, Winter JB, Pandey V, Sawaya
            MR, Wohlschlegel J, Loo JA, Rodriguez JA, Clarke SG
  TITLE     Structural basis for L-isoaspartyl-containing protein recognition by
            the PCMTD1 cullin-RING E3 ubiquitin ligase.
  JOURNAL   bioRxiv [Preprint]. 2025 May 21:2025.05.21.654933. doi: 
            10.1101/2025.05.21.654933.
  PUBMED    40475564
REFERENCE   2  (bases 1 to 6045)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 May 21:2025.05.21.654933. doi: 
            10.1101/2025.05.21.654933."
COMMENT     Sequence Label: pET-28a(+) PCMT1 H58 A214
FEATURES             Location/Qualifiers
     source          1..6045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             709..726
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(779..797)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      793..840
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      877..1332
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(964..983)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1174..1195
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1424..2239)
                     /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(1424..2239)
                     /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     2147..2166
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2231..2250
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2361..2949
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2850..2869
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3103..3120
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3135..3274
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3200..3219)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3360..3382
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3376..3567)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4376..5458)
                     /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     5420..5439
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5459..5536)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5742..5761
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5845..5864
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5845..5863
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5864..5888
                     /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             5903..5925
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5944..5961
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5971..5988
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5992..6024
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
ORIGIN
        1 atggcatgga aaagcggcgg ggccagtcac tcagaactta ttcataatct tcggaaaaac
       61 ggtataatta aaaccgataa ggtatttgag gtgatgctgg ctactgatcg gtctcactat
      121 gcgaaatgca atccatatat ggacagccct cagagcattg ggtttcaggc gcacatttca
      181 gcgccgcata tgcacgcata tgcactcgaa ctgctttttg accagctgca cgaaggagcg
      241 aaagctctgg atgtgggatc tgggagtggt attctgacgg catgtttcgc ccgaatggtg
      301 ggatgcaccg gtaaagtgat tggtattgac cacatcaagg aacttgtgga tgatagtgtt
      361 aataatgtgc gcaaggatga tcctacgctg ctgagttcag gtcgtgtgca attagtggtc
      421 ggggatggtc gaatgggcta tgcagaggaa gcaccatatg atgcgattca cgttggcgcg
      481 gccgccccag tggtcccaca ggccctgatt gaccagctga agcctggggg ccggctcatt
      541 ttacctgtag gcccggcagg aggcaaccaa atgttagaac agtacgacaa attgcaagat
      601 ggctctatca aaatgaaacc cttgatggga gtcatttacg ctccgctcac agataaggaa
      661 aaacaatggt ctagagatga gttgtgaaag cttgcggccg cactcgagca ccaccaccac
      721 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      781 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      841 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
      901 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      961 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1021 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1081 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1141 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1201 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1261 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1321 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1381 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     1441 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     1501 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     1561 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     1621 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     1681 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     1741 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     1801 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     1861 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     1921 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     1981 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     2041 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     2101 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     2161 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     2221 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     2281 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2341 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2401 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2461 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2521 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2581 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2641 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2701 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2761 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2821 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2881 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2941 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3001 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3061 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3121 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3181 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3241 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3301 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3361 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3421 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3481 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3541 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3601 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3661 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     3721 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     3781 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     3841 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     3901 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     3961 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4021 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     4081 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     4141 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4201 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     4261 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     4321 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     4381 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4441 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     4501 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     4561 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4621 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     4681 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     4741 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     4801 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     4861 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     4921 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     4981 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     5041 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5101 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     5161 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     5221 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     5281 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     5341 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     5401 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     5461 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     5521 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     5581 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     5641 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     5701 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     5761 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     5821 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
     5881 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac catgggcagc
     5941 agccatcatc atcatcatca cagcagcggc ctggtgccgc gcggcagcca tatggctagc
     6001 atgactggtg gacagcaaat gggtcgcgga tccgaattcg agctc
//
