LOCUS       sequence-7393-ex        6031 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6031)
  AUTHORS   Kim HW, Ahn HJ, Yoon HJ, Kim HW, Baek SH, Suh SW
  TITLE     Crystallization and preliminary X-ray crystallographic analysis of 
            tRNA(m1G37)methyltransferase from Haemophilus influenzae.
  JOURNAL   Acta Crystallogr D Biol Crystallogr. 2003 Jan . 59(Pt 1):183-4.
  PUBMED    12499565
REFERENCE   2  (bases 1 to 6031)
  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: "Acta 
            Crystallogr D Biol Crystallogr. 2003 Jan . 59(Pt 1):183-4."
COMMENT     Sequence Label: TrmD_HaeIn/pET28b(+) N,C-tag
FEATURES             Location/Qualifiers
     source          1..6031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(905..922)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(932..949)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             968..990
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1005..1029
                     /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(1029..1048)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1030..1048)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1132..1151)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1357..1434
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1435..2517
                     /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(1454..1473)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3326..3517
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3511..3533)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3619..3761
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3674..3693
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3776..3793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3947..4535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4027..4046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4646..4665)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4657..5472
                     /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             4657..5472
                     /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     complement(4730..4749)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5565..6020)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5702..5723)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5914..5933
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagactgtta tgctcggctt
      181 gcgcctcttt taacagctta cgttgttcgt cagtcagagc taggccttct aagagctcag
      241 ggcgtcggag ccacgttctc tgtagcgatt gttttaatcg ccatttccga atttcttcgt
      301 gatgtcccga catcagcacg ggtggtacag ttaatccttc taacacttct ggtctagtgt
      361 aatgcgggca atctaataaa ccatctgcaa aagaatcttc ttctgccgag gcttgtttgc
      421 ctaatacacc gggaataaaa cgtgcaacag catcaattaa tgtcattgcc ggcaattccc
      481 caccggtcag aacgtaatcg ccgattgacc attcttcatc aatttcagtt tgaatcaacc
      541 gttcatcaat cccttcgtaa cgtccacata ctaaaatcaa tttctgattt tgagcaagct
      601 cggttacgcc gccttgatcg agtttacgtc cttgtggcga aaggtaaatc acctttgcgc
      661 cttctcctgc cgcagctttt gcagtatgaa tcgcatcccg taaaggttgc accatcatta
      721 gcattcctgg gccaccacca taagggcgat cgtccacggt tttatgcttg tcgaatgtaa
      781 aatctcttgg attccaacat tcaactttca gaagattatg ttttacggct ctacctgtaa
      841 ccccaaattc cgtaattgct ttaaacattt cggggaataa tgaaattacc cctatccaca
      901 tatggctgcc gcgcggcacc aggccgctgc tgtgatgatg atgatgatgg ctgctgccca
      961 tggtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct
     1021 cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg
     1081 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     1141 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     1201 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     1261 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     1321 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     1381 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     1441 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     1501 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     1561 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     1621 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     1681 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     1741 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     1801 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     1861 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     1921 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     1981 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     2041 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     2101 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     2161 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     2221 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     2281 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     2341 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     2401 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     2461 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     2521 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
     2581 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     2641 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
     2701 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
     2761 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
     2821 cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct
     2881 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     2941 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     3001 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     3061 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     3121 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     3181 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     3241 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     3301 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     3361 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     3421 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
     3481 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3541 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3601 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     3661 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat atgcggtgtg
     3721 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     3781 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3841 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3901 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3961 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4021 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4081 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4141 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4201 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4261 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4321 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4381 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4441 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4501 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4561 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa
     4621 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     4681 tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     4741 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     4801 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     4861 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     4921 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag
     4981 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     5041 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     5101 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     5161 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     5221 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     5281 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     5341 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     5401 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     5461 gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5521 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaaattg taaacgttaa
     5581 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5641 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5701 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     5761 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     5821 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     5881 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     5941 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     6001 tgcgccgcta cagggcgcgt cccattcgcc a
//
