LOCUS       sequence-232467-        6553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express TS2126 RNA ligase in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6553)
  AUTHORS   Miura F, Shibata Y, Miura M, Sangatsuda Y, Hisano O, Araki H, Ito T
  TITLE     Highly efficient single-stranded DNA ligation technique improves 
            low-input whole-genome bisulfite sequencing by post-bisulfite 
            adaptor tagging.
  JOURNAL   Nucleic Acids Res. 2019 May 22. pii: 5494777. doi: 
            10.1093/nar/gkz435.
  PUBMED    31114914
REFERENCE   2  (bases 1 to 6553)
  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: "Nucleic 
            Acids Res. 2019 May 22. pii: 5494777. doi: 10.1093/nar/gkz435."
COMMENT     Sequence Label: pET28a-TS2126 RNA Ligase
FEATURES             Location/Qualifiers
     source          1..6553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             148..180
                     /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"
     CDS             1418..1435
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1488..1506)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1502..1549
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1586..2041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1673..1692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1883..1904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2133..2948)
                     /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(2133..2948)
                     /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     2856..2875
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2940..2959
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3070..3658
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3559..3578
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3812..3829
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3844..3983
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3909..3928)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4069..4091
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4085..4276)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5085..6167)
                     /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     6129..6148
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6168..6245)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6451..6470
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcatga ctggtggaca gcaaatgggt
      181 cgcggatcca tgagctcact ggctccgtgg cgtacgacga gctggagtcc gctgggctct
      241 ccgccaagtt tagaggatgc tttgcgtctt gcgcgcacaa ctcgcgcatt cgcagtccgc
      301 cgcgatggtg aaggtcgcgc attggttacc tacctgtatg gcactcccga gctgttctcc
      361 ctgccgggcg cgcgtgaatt gcgtggtatc gtgtatcgcg aggaggatgg caccgtgctg
      421 agccgtccgt ttcacaaatt cttcaacttt ggagaaccgt tagctccggg tgaagaggcc
      481 tttaaagcat ttcgcgattc gatggtgccc ctgtttgtcg ccgagaaagt ggatggctac
      541 ctggcacaag cgtacctgga tggtggggag ttgcgttttg cctctcggca tagccttaat
      601 ccgccacttg tgggtgcgtt gctgcgcaaa gccgtcgatg aagaagcgat ggcgcgtctg
      661 ggaaaactct tagctgcgga aggcgggcgt tggacggccc tgttagaagt ggttgatccg
      721 gaagcgccgg tcatggtacc gtatcaggaa ccaggcgtgt atctgctggc cctccgttcg
      781 attggtgaag ggcactatct tctgcctggg gtacatttcc cgctgcctga agccctgcgt
      841 tacgttcggt gggaaccacg catggacttt gaccctcatc gctttcgcgg tgaaattcgc
      901 gacctccaag gcgtagaggg ctacgtggtt accgatggtg cggagtttgt caagttcaaa
      961 accggctggg cgtttcggtt agcgcgcttc ctgatggacc ccgaaggggt gttcctggaa
     1021 gcctatgcgg aagatcggct ggacgacctg gtgggtgcct tggcgggccg cgaggacctc
     1081 ctgcgtgcgg ttgcgcgtgc gcaggattac ctggcaggac tctatggtga agcagttgga
     1141 gctggcgatg ccttacgccg tatgggcctt ccgcgcaagg aagcatgggc gcgtgtacag
     1201 gaagaggccg gtcgttgggg cggctttgcc cctgcgtatg ctcgcgcagc aatggccgca
     1261 tatgaaggcg gcgaagcccg cgaagcgttt ctggtcgaac tgcgcaaacg ctccgctcgt
     1321 aaagctctgg aagctctgca cttgttccca cgcgttggtg gggaattacg cggttaagaa
     1381 ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga
     1441 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     1501 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     1561 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     1621 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     1681 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     1741 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     1801 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1861 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1921 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1981 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     2041 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2101 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     2161 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     2221 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     2281 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     2341 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     2401 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     2461 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     2521 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     2581 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     2641 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     2701 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     2761 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     2821 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     2881 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     2941 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     3001 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3061 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3121 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3181 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3241 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3301 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3361 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3421 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3481 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3541 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3601 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3661 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3721 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3781 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3841 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     3901 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     3961 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     4021 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4081 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     4141 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     4201 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     4261 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     4321 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     4381 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     4441 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     4501 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     4561 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     4621 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     4681 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     4741 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     4801 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     4861 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     4921 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     4981 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     5041 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     5101 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5161 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     5221 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     5281 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     5341 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     5401 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     5461 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5521 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5581 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5641 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5701 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5761 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5821 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5881 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5941 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     6001 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     6061 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     6121 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     6181 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     6241 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     6301 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     6361 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     6421 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     6481 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     6541 gatcccgcga aat
//
