LOCUS       sequence-324518-        6508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express TS2126 RNA ligase with His6 tag and Strep tag at N and C 
            terminal, respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6508)
  AUTHORS   Hisano O, Ito T, Miura F
  TITLE     Short single-stranded DNAs with putative non-canonical structures 
            comprise a new class of plasma cell-free DNA.
  JOURNAL   BMC Biol. 2021 Oct 14;19(1):225. doi: 10.1186/s12915-021-01160-8.
  PUBMED    34649537
REFERENCE   2  (bases 1 to 6508)
  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: "BMC Biol.";
            date: "2021-10-14"; volume: "19(1):225. doi"; pages: " 
            10.1186/s12915-021-01160-8"
COMMENT     Sequence Label: pET28a-TS2126 RnlA-Strep
FEATURES             Location/Qualifiers
     source          1..6508
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     922..941
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(961..1038)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1244..1263
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1347..1366
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1347..1365
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1366..1390
                     /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             1405..1427
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1446..1463
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1473..1490
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1494..1526
                     /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             2727..2750
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(2789..2807)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2803..2850
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2887..3342
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2974..2993)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3184..3205
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3434..4249)
                     /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(3434..4249)
                     /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     4157..4176
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4241..4260
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4371..4959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4860..4879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5113..5130
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5145..5284
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5210..5229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5370..5392
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5386..5577)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6386..6508,1..960))
                     /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"
ORIGIN
        1 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
       61 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
      121 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
      181 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
      241 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
      301 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
      361 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
      421 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
      481 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
      541 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
      601 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
      661 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
      721 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
      781 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
      841 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
      901 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
      961 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1021 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1081 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     1141 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     1201 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     1261 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     1321 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     1381 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggca
     1441 gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatggcta
     1501 gcatgactgg tggacagcaa atgggtcgcg gatccatgag ctcactggct ccgtggcgta
     1561 cgacgagctg gagtccgctg ggctctccgc caagtttaga ggatgctttg cgtcttgcgc
     1621 gcacaactcg cgcattcgca gtccgccgcg atggtgaagg tcgcgcattg gttacctacc
     1681 tgtatggcac tcccgagctg ttctccctgc cgggcgcgcg tgaattgcgt ggtatcgtgt
     1741 atcgcgagga ggatggcacc gtgctgagcc gtccgtttca caaattcttc aactttggag
     1801 aaccgttagc tccgggtgaa gaggccttta aagcatttcg cgattcgatg gtgcccctgt
     1861 ttgtcgccga gaaagtggat ggctacctgg cacaagcgta cctggatggt ggggagttgc
     1921 gttttgcctc tcggcatagc cttaatccgc cacttgtggg tgcgttgctg cgcaaagccg
     1981 tcgatgaaga agcgatggcg cgtctgggaa aactcttagc tgcggaaggc gggcgttgga
     2041 cggccctgtt agaagtggtt gatccggaag cgccggtcat ggtaccgtat caggaaccag
     2101 gcgtgtatct gctggccctc cgttcgattg gtgaagggca ctatcttctg cctggggtac
     2161 atttcccgct gcctgaagcc ctgcgttacg ttcggtggga accacgcatg gactttgacc
     2221 ctcatcgctt tcgcggtgaa attcgcgacc tccaaggcgt agagggctac gtggttaccg
     2281 atggtgcgga gtttgtcaag ttcaaaaccg gctgggcgtt tcggttagcg cgcttcctga
     2341 tggaccccga aggggtgttc ctggaagcct atgcggaaga tcggctggac gacctggtgg
     2401 gtgccttggc gggccgcgag gacctcctgc gtgcggttgc gcgtgcgcag gattacctgg
     2461 caggactcta tggtgaagca gttggagctg gcgatgcctt acgccgtatg ggccttccgc
     2521 gcaaggaagc atgggcgcgt gtacaggaag aggccggtcg ttggggcggc tttgcccctg
     2581 cgtatgctcg cgcagcaatg gccgcatatg aaggcggcga agcccgcgaa gcgtttctgg
     2641 tcgaactgcg caaacgctcc gctcgtaaag ctctggaagc tctgcacttg ttcccacgcg
     2701 ttggtgggga attacgcggt gaattctgga gccatccgca gtttgaaaaa taacaaagcc
     2761 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2821 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     2881 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2941 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3001 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3061 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3121 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3181 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3241 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3301 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     3361 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3421 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     3481 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3541 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     3601 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     3661 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     3721 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     3781 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     3841 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     3901 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     3961 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     4021 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     4081 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     4141 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     4201 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     4261 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     4321 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4381 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4441 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4501 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4561 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4621 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4681 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4741 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4801 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4861 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4921 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4981 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5041 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5101 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     5161 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     5221 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5281 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5341 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5401 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5461 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5521 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5581 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5641 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5701 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5761 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5821 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5881 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     5941 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     6001 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     6061 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     6121 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     6181 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     6241 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     6301 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6361 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6421 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     6481 gggtggtttt tcttttcacc agtgagac
//
