LOCUS       sequence-451307-        6642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of MYPT1(1-314).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6642)
  AUTHORS   Claes Z, Bollen M
  TITLE     A split-luciferase lysate-based approach to identify small-molecule 
            modulators of phosphatase subunit interactions.
  JOURNAL   Cell Chem Biol. 2023 Dec 21;30(12):1666-1679.e6. doi: 
            10.1016/j.chembiol.2023.07.018. Epub 2023 Aug 24.
  PUBMED    37625414
REFERENCE   2  (bases 1 to 6642)
  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.chembiol.2023.07.018"; journalName: "Cell Chem Biol"; 
            date: "2023-12-21- 21"; volume: "30"; issue: "12"; pages: 
            "1666-1679.e6"
COMMENT     Sequence Label: pET16b-MYPT1(1-314)
FEATURES             Location/Qualifiers
     source          1..6642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1270..1281)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(1294..1323)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1336..1358
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1373..1397
                     /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(1397..1416)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1398..1416)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1500..1519)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1725..1802
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1803..2885
                     /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(1822..1841)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4197..4388
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4382..4404)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4490..4629
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4545..4564
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4644..4661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4815..5403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4895..4914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5574..6365,6366..6434))
                     /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     6197..6216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6435..6539)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6607..6625
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atcctcactt ctgtgactga ttattgtcca tatttgctgt
      361 tgattcaatt agtggagatt tcttgtcccg tttttcacta tggagcagat tttgtttctt
      421 ttgcaactct tctaaatatc ctaaaatgtc ttcatctgct acatcaaagg ctgtttggcc
      481 cactttgttg accatctcca tatcacacag attgtccact aaaattcgac atgcttcttc
      541 tttaccccaa tgagctgcag catgaagagg tgtccagcca tcatagtctt taatattaac
      601 atcatagcct gcctgtatta aaagttttaa aacttccgta tagcctttag cagctgcaac
      661 gtgaagtgct gtacctccag attttgcatg ccggacatca tttatatgac cactatttag
      721 ccactgcctg gcatctctaa gcatgatccg ttcttcttcc tttcgagctg cttctatatc
      781 aaccccttgc cgattaactt cattttgaag tagctcttcc attgcctcct cctccgcaat
      841 atctaaaggt gtatctcctt cactgttgac agcccctaca tgtgctcctt gaccaatcaa
      901 aaactctgca atatcaagat atccacagga agctgctgca tgtagtggta tccagccttc
      961 attatcaggt tgattaatat ttgctccatt ttctaccaga aacttcacca tatcaacatt
     1021 gtcatcaatg caagcctggt gcagggcagt gagtccgtcc acattggcgt aattgatgtc
     1081 ggcgccgcgg tgcagcagct tgaggacctc gtccgtgtcg ccgctggagc aagcagccag
     1141 gaagacggcg ccatcgtcga acttcacctt ggtcttctgg cgcttcacca ccggaggctc
     1201 gaggtccgtc tcggagccga tccagcgttt cagctgctcg ttccgcttct gcttcgcgtc
     1261 cgccatatga cgaccttcga tatggccgct gctgtgatga tgatgatgat gatgatgatg
     1321 atggcccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
     1381 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat
     1441 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
     1501 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
     1561 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
     1621 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
     1681 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
     1741 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     1801 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     1861 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     1921 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     1981 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     2041 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     2101 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     2161 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     2221 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     2281 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     2341 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     2401 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     2461 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     2521 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     2581 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     2641 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     2701 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     2761 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     2821 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2881 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
     2941 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     3001 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     3061 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
     3121 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc
     3181 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac
     3241 gtcttgctgg cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct
     3301 tccggcggca tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac
     3361 catcagggac agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcattgga
     3421 ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg
     3481 attgtaggcg ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc
     3541 cgggccacct cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa
     3601 gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga
     3661 acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg
     3721 ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     3781 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     3841 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     3901 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg
     3961 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
     4021 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac
     4081 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc
     4141 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga
     4201 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
     4261 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
     4321 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
     4381 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     4441 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     4501 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
     4561 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4621 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4681 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4741 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4801 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4861 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4921 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4981 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5041 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5101 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5161 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5221 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     5281 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5341 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5401 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5461 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5521 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5581 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5641 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5701 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5761 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5821 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5881 gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5941 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6001 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6061 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6121 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6181 ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6241 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6301 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6361 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6421 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6481 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6541 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     6601 tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aa
//
