LOCUS       sequence-353282-        6630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for production of recombinant T. terrestris CutA 
            NTase fragment in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6630)
  AUTHORS   Tomecki R, Kobylecki K, Drazkowska K, Hyjek-Skladanowska M, 
            Dziembowski A
  TITLE     Reproducible and efficient new method of RNA 3'-end labelling by 
            CutA nucleotidyltransferase-mediated CC-tailing.
  JOURNAL   RNA Biol. 2021 Nov 12;18(sup2):623-639. doi: 
            10.1080/15476286.2021.1999104. Epub 2021 Nov 12.
  PUBMED    34766865
REFERENCE   2  (bases 1 to 6630)
  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"; journalName: "RNA Biol.";
            date: "2021-11-12"; volume: "18(sup2):623-639. doi"; pages: " 
            10.1080/15476286.2021.1999104. Epub 2021 Nov 12"
COMMENT     Sequence Label: pCC25
FEATURES             Location/Qualifiers
     source          1..6630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..34)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      30..77
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      114..569
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(201..220)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     411..432
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(661..1476)
                     /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(661..1476)
                     /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     1384..1403
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1468..1487
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1598..2186
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2087..2106
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2340..2357
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2372..2511
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2437..2456)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2597..2619
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2613..2804)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3613..4695)
                     /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     4657..4676
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4696..4773)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4979..4998
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5082..5101
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5082..5100
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5101..5125
                     /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             5140..5162
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5181..5198
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5208..5225
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6576..6593
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
       61 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
      121 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      181 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      241 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      301 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
      361 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
      421 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
      481 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
      541 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
      601 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
      661 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
      721 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
      781 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
      841 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
      901 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
      961 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1021 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     1081 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     1141 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     1201 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     1261 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     1321 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     1381 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     1441 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     1501 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     1561 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1621 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1681 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1741 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1801 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1861 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1921 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1981 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2041 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2101 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2161 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2221 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2281 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2341 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2401 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     2461 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     2521 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     2581 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     2641 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     2701 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     2761 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     2821 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     2881 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     2941 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3001 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3061 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3121 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3181 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     3241 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     3301 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     3361 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     3421 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     3481 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     3541 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     3601 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3661 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     3721 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     3781 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     3841 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     3901 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     3961 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     4021 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     4081 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     4141 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     4201 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     4261 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     4321 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     4381 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     4441 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     4501 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     4561 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     4621 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     4681 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     4741 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     4801 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     4861 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     4921 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     4981 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     5041 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     5101 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     5161 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg
     5221 gcagccatat ggaaggtgaa tatattaaac tcaaagtcat tggacaggat agcagtgaga
     5281 ttcacttcaa agtgaaaatg acaacacatc tcaagaaact caaagaatca tactgtcaaa
     5341 gacagggtgt tccaatgaat tcactcaggt ttctctttga gggtcagaga attgctgata
     5401 atcatactcc aaaagaactg ggaatggagg aagaagatgt gattgaagtc taccaggaac
     5461 aaaccggtgg atccttcgaa acggcaattc cgtttagccg tcgccgtatg ccgtactctc
     5521 tgggcaccga taagctggaa aaagtggatc cggacaaaat caagagtaaa ctgtccgaag
     5581 acgttgaacg taaactggaa acggatatgc gtgaactgta tgaccgcctg ctgccgaccg
     5641 aagcgattga agttaaccgc cgtgaactgg tcagcaaact ggaacgcctg ttcaataccg
     5701 aatggccggg tcatgatatc cgtgttcacc tgtttggtag ttccggcaat ctgctgtgct
     5761 cagatgactc ggatgttgac atttgtatca ccacgccgtg gcgcgaactg gaaagcgtct
     5821 gcatgattgc agaactgctg gatcgtcatg gcatggaaaa ggtggtttgt gtgtcatcgg
     5881 cgaaggtgcc gattgttaaa atctgggatc cggaactgaa actggcatgc gacatgaacg
     5941 ttaacaatac cctggctctg gaaaataccc gcatggtccg tacgtatgtg agcattgatg
     6001 accgcgtgcg tccgctggcg atgattatca aatactggac ccgccgtcgc gtcgtgaacg
     6061 atgcagcctt cggtggcacc ctgagctctt acacgtggat ctgtatgatt atcgcatttc
     6121 tgcagctgcg tgatccgccg gtcctgccgg ctctgcatca gcaacacgac ctgaagctgg
     6181 tgaaacagga tggtgcgctg tctgacttcg ccgatgacat tccgaaactg cgtggttttg
     6241 gcgcgaagaa taaagatagt ctggccgtgc tgctgtttca gtttttccgc ttctatgcac
     6301 acgaatttga ttatgacaaa tacaccctga gcatccgtat gggcaccctg ctgacgaagg
     6361 cagaaaagaa ctggcagtac ctggtgaaca atgctctgtg cgttgaagaa ccgtttaatg
     6421 atggtcgcaa cctgggcaat accgccgacg aaacgagctt ccgtggtctg catatggaac
     6481 tgcgtcgcgc atttgatctg attgctgaag gcaaactgga agaatgctgt gaacagtatg
     6541 tgtttccgaa agaagaagaa cgcgtttaac tcgagcacca ccaccaccac cactgagatc
     6601 cggctgctaa caaagcccga aaggaagctg
//
