LOCUS       sequence-9589-ex        5622 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 5622)
  AUTHORS   Hu H, Clarkson MW, Hermans J, Lee AL
  TITLE     Increased rigidity of eglin c at acidic pH: evidence from NMR spin 
            relaxation and MD simulations.
  JOURNAL   Biochemistry. 2003 Dec 2. 42(47):13856-68.
  PUBMED    14636053
REFERENCE   2  (bases 1 to 5622)
  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: 
            "Biochemistry. 2003 Dec 2. 42(47):13856-68."
COMMENT     Sequence Label: pET28a-EgC*
FEATURES             Location/Qualifiers
     source          1..5622
                     /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"
     primer_bind     complement(620..639)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(621..639)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(723..742)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        948..1025
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1026..2108
                     /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(1045..1064)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2917..3108
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3102..3124)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3210..3352
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3265..3284
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3367..3384)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3538..4126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3618..3637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4237..4256)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4248..5063
                     /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             4248..5063
                     /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(4321..4340)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5156..5611)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5293..5314)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5505..5524
                     /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 cgaattcgcc acacgcgaac tttacggtca tcgcgtggtg cacaactgat
      241 cacggtttga taatcaatcg cgcagggcag aacgaaaccg tcgttgtagt cggtgtgttc
      301 accaatcaaa ttcacgcggc caggcgatta attaattact aacctacgtg aggaacatga
      361 ttaacaacgt tagtacctgg gttgtagaaa acgcgtacac ggttgtaacg aaggtccaga
      421 gtaacaggag aaccttccgg caggaagtag acgtcgtatt gtgggtaatg cagagtgaag
      481 tattcacgag cctggtcgac agttttacca acaacctcgg gccaagactt aagttcagaa
      541 ccaaattcag tcatggtata tccttcttaa agttaaacaa aattatttct agaggggaat
      601 tgttagccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
      661 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
      721 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
      781 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
      841 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
      901 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
      961 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     1021 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     1081 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     1141 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     1201 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     1261 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     1321 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     1381 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     1441 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     1501 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     1561 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     1621 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     1681 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     1741 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     1801 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     1861 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     1921 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     1981 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     2041 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     2101 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     2161 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     2221 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     2281 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     2341 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     2401 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc
     2461 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     2521 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     2581 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     2641 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     2701 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     2761 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     2821 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     2881 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     2941 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     3001 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     3061 taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3121 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3181 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     3241 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     3301 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     3361 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3421 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3481 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3541 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3601 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3661 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3721 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3781 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3841 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3901 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3961 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4021 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4081 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4141 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4201 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     4261 aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg
     4321 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
     4381 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     4441 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     4501 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa
     4561 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     4621 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     4681 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     4741 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     4801 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     4861 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     4921 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     4981 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     5041 tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt
     5101 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt
     5161 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5221 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5281 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5341 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5401 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     5461 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5521 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5581 gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca
//
