LOCUS       sequence-357004-        5927 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 5927)
  AUTHORS   Huth JR, Bewley CA, Jackson BM, Hinnebusch AG, Clore GM, Gronenborn 
            AM
  TITLE     Design of an expression system for detecting folded protein domains 
            and mapping macromolecular interactions by NMR.
  JOURNAL   Protein Sci. 1997 Nov . 6(11):2359-64.
  PUBMED    9385638
REFERENCE   2  (bases 1 to 5927)
  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: "Protein 
            Sci. 1997 Nov . 6(11):2359-64."
COMMENT     Sequence Label: GEV 1
FEATURES             Location/Qualifiers
     source          1..5927
                     /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             95..256
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLALNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             659..676
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(729..747)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      743..790
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      827..1282
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(914..933)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1124..1145
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1308..1412
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1413..1481,1482..2273)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1631..1650)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2444..3032
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2933..2952
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3186..3203
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3218..3357
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3283..3302)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3443..3465
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3459..3650)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4459..5541)
                     /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     5503..5522
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5542..5619)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5825..5844
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcagtacaag cttgctctga acggtaaaac
      121 cctgaaaggt gaaaccacca ccgaagctgt tgacgctgct accgcggaaa aagttttcaa
      181 acagtacgct aacgacaacg gtgttgacgg tgaatggacc tacgacgacg ctaccaaaac
      241 cttcacggta accgaaccgg gcggtccggc tagcgcgctc agccctgtgc cacctgtggt
      301 ccatctgacc ctccgccggg ctggggatga cttctctcgt cgctaccgtc gtgacttcgc
      361 agagatgtcc agtcagctgc acctgacgcc cttcaccgcg aggggacgct ttgccacggt
      421 ggtggaggaa ctcttcaggg atggggtgaa ctgggggagg attgtggcct tctttgagtt
      481 cggtggggtc atgtgtgtgg agagcgtcaa cagggagatg tcacccctgg tggacaacat
      541 cgccctgtgg atgactgagt acctgaaccg gcatctgcac acctggatcc aggataacgg
      601 aggctgggat gcctttgtgg aactatatgg ccccagcatg cgaaaaaagt aactcgagca
      661 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
      721 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
      781 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
      841 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
      901 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
      961 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1021 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1081 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1141 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1201 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1261 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1321 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1381 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1441 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1501 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1561 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1621 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1681 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1741 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1801 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1861 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1921 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     1981 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2041 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2101 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2161 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2221 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2281 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2341 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2401 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2461 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2521 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2581 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2641 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2701 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2761 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2821 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2881 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2941 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3001 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3061 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3121 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3181 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3241 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     3301 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3361 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3421 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3481 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3541 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3601 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3661 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3721 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     3781 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3841 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3901 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3961 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4021 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4081 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4141 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4201 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4261 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4321 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4381 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4441 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4501 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4561 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4621 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4681 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4741 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4801 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4861 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4921 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4981 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5041 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5101 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5161 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5221 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5281 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5341 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5401 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5461 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5521 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5581 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5641 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5701 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5761 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5821 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5881 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
