LOCUS       sequence-443740-        5601 bp    DNA     circular SYN 15-APR-2025
DEFINITION  A universal plasmid for expressing any protein with TetraCysteine 
            motif.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5601)
  AUTHORS   Antonenko A, Pomorski A, Singh AK, Kapczynska K, Krezel A
  TITLE     Site-Specific and Fluorescently Enhanced Installation of 
            Post-Translational Protein Modifications via Bifunctional 
            Biarsenical Linker.
  JOURNAL   ACS Omega. 2024 Oct 30;9(45):45127-45137. doi: 
            10.1021/acsomega.4c05828. eCollection 2024 Nov 12.
  PUBMED    39554417
REFERENCE   2  (bases 1 to 5601)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acsomega.4c05828"; journalName: "ACS Omega"; date: 
            "2024-10-30- 30"; volume: "9"; issue: "45"; pages: "45127-45137"
COMMENT     Sequence Label: TCT-Universal plasmid
FEATURES             Location/Qualifiers
     source          1..5601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..45
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     110..129
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      240..828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     729..748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     982..999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1014..1153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1079..1098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1239..1261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1255..1446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2255..3337)
                     /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     3299..3318
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3338..3415)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3621..3640
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3724..3743
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3724..3742
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3743..3767
                     /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             3782..3804
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3823..3840
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3850..3867
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3874..3894
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3910..3927
                     /codon_start=1
                     /product="tetracysteine peptide that binds biarsenical 
                     labeling reagents"
                     /label=tetracysteine tag
                     /translation="CCPGCC"
     CDS             4189..4206
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4259..4277)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4273..4320
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4357..4812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4444..4463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4654..4675
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4904..5601,1..118))
                     /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(join(4904..5601,1..118))
                     /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"
ORIGIN
        1 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
       61 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
      121 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
      181 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
      241 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
      301 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
      361 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
      421 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      481 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      541 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      601 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      661 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      721 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      781 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      841 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
      901 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
      961 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     1021 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     1081 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     1141 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     1201 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     1261 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     1321 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     1381 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     1441 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     1501 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     1561 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     1621 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     1681 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     1741 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     1801 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     1861 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     1921 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     1981 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     2041 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     2101 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     2161 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     2221 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2281 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2341 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     2401 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     2461 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     2521 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     2581 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     2641 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     2701 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     2761 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     2821 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     2881 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     2941 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     3001 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     3061 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     3121 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     3181 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     3241 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     3301 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     3361 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     3421 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     3481 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     3541 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     3601 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     3661 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     3721 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     3781 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
     3841 agcagcggcc tggtgccgcg cggcagccat atggaaaacc tgtattttca gggcgtggaa
     3901 ttcctgaact gctgtccggg ctgctgtatg gaaccgggat ccctgccgga aaccggcatg
     3961 gacccggaaa cctgcccgtg cccgtctggt ggctcctgta cctgtgccga ttcgtgtaaa
     4021 tgtgaaggct gtaaatgtac ctcatgcaaa aagagctgct gttcttgctg tccggcggaa
     4081 tgcgaaaaat gtgcaaagga ttgcgtgtgt aaaggcggtg aagcggccga agcagaagct
     4141 gaaaagtgta gttgctgtca gtaaaagctt ccaaacacag gtctcgagca ccaccaccac
     4201 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     4261 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     4321 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     4381 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4441 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4501 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4561 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4621 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4681 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     4741 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4801 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4861 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     4921 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     4981 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     5041 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     5101 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     5161 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     5221 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     5281 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     5341 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     5401 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     5461 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     5521 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     5581 gcttcccata caatcgatag a
//
