LOCUS       sequence-7435-ex        6113 bp    DNA     circular SYN 15-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6113)
  AUTHORS   Yang JK, Park MS, Waldo GS, Suh SW
  TITLE     Directed evolution approach to a structural genomics project: Rv2002
            from Mycobacterium tuberculosis.
  JOURNAL   Proc Natl Acad Sci U S A. 2003 Jan 21. 100(2):455-60.
  PUBMED    12524453
REFERENCE   2  (bases 1 to 6113)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2003 Jan 21. 100(2):455-60."
COMMENT     Sequence Label: Rv2002 M3/pET-28b(+)N-tag
FEATURES             Location/Qualifiers
     source          1..6113
                     /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(987..1004)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1014..1031)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1050..1072
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1087..1111
                     /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(1111..1130)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1112..1130)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1214..1233)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1439..1516
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1517..2599
                     /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(1536..1555)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3408..3599
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3593..3615)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3701..3843
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3756..3775
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3858..3875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4029..4617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4109..4128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4728..4747)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4739..5554
                     /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             4739..5554
                     /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(4812..4831)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5647..6102)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5784..5805)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5996..6015
                     /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 tcccgtcacc cattccggct gcgaggacac ctcgacggca
      241 ccgaagtcgt tgtgtgccag gccagctacg gtcccgccgt cgaccacaaa ttccgcgccg
      301 gtagaatagc tcgactcatc gctggccagg tagacgacga ggttggacac ttccacgggt
      361 tcggccgcgc ggcccagcgc ggtctggaag atgtcttcgg ggacccagtc agtcatcggc
      421 gtcttgacca acccagggtg aatcgagttg actcgaattc cgctgggccc caactcgaga
      481 gcggtggact tggtcagccc ccgcacggcg aacttggtgg cggtatagcc atgacaagca
      541 accgtgccgg ccagcccctc gatcgacgaa atgttgatga tggagccgcg accagcctct
      601 ttcattggct tgacgacagc gcggatgccc aggaagactc cggtcaggtt gacatcgagg
      661 atgcgctgcc attcggtgag ggcgtagtcc tcgatcgtcc cgatgttgag aatgccggcg
      721 ttgttgacca gcacgtgcag gccaccgaat gcggtgaccg cggtgtccac cgcagccttc
      781 cattgcgcgg gttgggtaac gtcgagatgg acgtagcggg ccgcatcggc cagttcggcg
      841 gccatcgcct tgccctcctc gtcgaggatg tcgccgaaca caacctttgc gccttcggcc
      901 accatcgccc gcacatggga tgcacccata ccgcgcgccc cgccgctgac aagtgcgacc
      961 tttcctgtca accgtccaga catatggctg ccgcgcggca ccaggccgct gctgtgatga
     1021 tgatgatgat ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc
     1081 tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg
     1141 atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     1201 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
     1261 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
     1321 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
     1381 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga
     1441 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     1501 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     1561 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     1621 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     1681 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     1741 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     1801 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     1861 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     1921 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     1981 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     2041 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     2101 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     2161 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     2221 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     2281 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     2341 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     2401 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     2461 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     2521 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     2581 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     2641 cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     2701 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     2761 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
     2821 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
     2881 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc
     2941 cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
     3001 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
     3061 aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc
     3121 tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
     3181 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
     3241 tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca
     3301 gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg
     3361 gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg
     3421 aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga
     3481 aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg
     3541 ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     3601 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3661 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     3721 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     3781 agtgcaccat atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3841 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3901 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3961 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4021 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4081 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4141 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4201 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4261 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4321 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4381 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4441 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4501 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4561 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4621 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4681 attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     4741 gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc
     4801 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     4861 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     4921 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     4981 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     5041 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     5101 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     5161 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     5221 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     5281 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     5341 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     5401 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     5461 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     5521 gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat
     5581 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5641 cacctgaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     5701 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     5761 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     5821 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     5881 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     5941 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     6001 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     6061 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca
//
