LOCUS       sequence-237097-        5845 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 5845)
  AUTHORS   Chen S, Shin DH, Pufan R, Kim R, Kim SH
  TITLE     Crystal structure of methenyltetrahydrofolate synthetase from 
            Mycoplasma pneumoniae (GI: 13508087) at 2.2 A resolution.
  JOURNAL   Proteins. 2004 Sep 1. 56(4):839-43.
  PUBMED    15281135
REFERENCE   2  (bases 1 to 5845)
  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: "Proteins. 
            2004 Sep 1. 56(4):839-43."
COMMENT     Sequence Label: pSKB3.MPN348
FEATURES             Location/Qualifiers
     source          1..5845
                     /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(708..728)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(750..767)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             786..808
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    823..847
                     /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(847..866)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(848..866)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(950..969)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1175..1252
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1253..2335
                     /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(1272..1291)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3144..3335
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3329..3351)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3437..3576
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3492..3511
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3591..3608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3762..4350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3842..3861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4461..4480)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4472..5287
                     /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             4472..5287
                     /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(4545..4564)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5379..5834)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5516..5537)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5728..5747
                     /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 gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccttactcg agttcatcat taataattaa gtctagttgt
      241 acatcccaag gatcggctaa aaaatcacct ttttggaaac tgtacgctat tcctatttta
      301 ggttgttgtc tagttaattg cattaaataa cggtcataat agcccttgcc aaagcctaga
      361 cggtaattgt ctttattaaa gcctaccaat ggtattagga agcagtctat ttgattaggt
      421 tcgaatgtat ttgtctcctg atcaataaag ataatttcgg tatcacttat taccttgggg
      481 taaacagctc ttatttggtt aatctttaaa aactcaaaga agaagtcaac aaaagtaacc
      541 tcatttttaa tgggttcata aagtgcaatt gttttaatgc atggcttagg agttaaaaaa
      601 gcaactaatt tttgattaat cttttgatct aagtgacttt tttcaatggt acttaatgcc
      661 attctttttt gcagaatttg ttttcttaag gcatttttgt ccatatggcc ctggaagtaa
      721 aggttttcgg tcgttgggat atcgtaatcg tgatgatgat gatgatggct gctgcccatg
      781 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
      841 caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc
      901 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
      961 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
     1021 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
     1081 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
     1141 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
     1201 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     1261 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     1321 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     1381 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     1441 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     1501 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     1561 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     1621 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     1681 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     1741 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     1801 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     1861 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     1921 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     1981 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     2041 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     2101 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     2161 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     2221 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2281 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2341 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     2401 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     2461 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     2521 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     2581 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     2641 gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc
     2701 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     2761 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     2821 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     2881 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     2941 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     3001 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     3061 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     3121 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     3181 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     3241 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     3301 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     3361 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     3421 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     3481 gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata
     3541 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     3601 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3661 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3721 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3781 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3841 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3901 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3961 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4021 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4081 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4141 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4201 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4261 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4321 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4381 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc
     4441 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg
     4501 ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     4561 cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc
     4621 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     4681 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     4741 tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt
     4801 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     4861 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     4921 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     4981 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     5041 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     5101 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     5161 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     5221 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     5281 tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5341 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     5401 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     5461 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     5521 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     5581 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     5641 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     5701 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     5761 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     5821 gctacagggc gcgtcccatt cgcca
//
