LOCUS       sequence-315352-        5833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GB1 WT double mutant (A34F/V39L).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5833)
  AUTHORS   Damry AM, Mayer MM, Broom A, Goto NK, Chica RA
  TITLE     Origin of conformational dynamics in a globular protein.
  JOURNAL   Commun Biol. 2019 Nov 26;2:433. doi: 10.1038/s42003-019-0681-2. 
            eCollection 2019.
  PUBMED    31799435
REFERENCE   2  (bases 1 to 5833)
  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: "Commun 
            Biol."; date: "2019-11-26"; volume: "2:433. doi"; pages: " 
            10.1038/s42003-019-0681-2. eCollection 2019"
COMMENT     Sequence Label: pET-11a (GB1 WT - A34F/V39L)
FEATURES             Location/Qualifiers
     source          1..5833
                     /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             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(334..352)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      348..395
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(570..575,576..592,593..598))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(625..643)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        711..815
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(816..884,885..1676)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1034..1053)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1847..2435
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2336..2355
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2589..2606
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2621..2760
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2686..2705)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2846..2868
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2862..3053)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4365..5447)
                     /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     5409..5428
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5448..5525)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5731..5750
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcatcaccat caccatcacg gtatgactta
      121 caaattaatc cttaatggta aaacattgaa aggcgaaaca actactgaag ctgttgatgc
      181 tgctactgca gaaaaagtct tcaaacaata ctttaacgac aacggtcttg acggtgaatg
      241 gacttacgac gatgcgacta agacctttac agttactgaa tgaggatccg gctgctaaca
      301 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
      361 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
      421 atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt
      481 gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg
      541 ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa gctgtcaaac
      601 atgagaattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
      661 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
      721 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
      781 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
      841 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
      901 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
      961 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1021 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     1081 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1141 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1201 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1261 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1321 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     1381 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1441 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1501 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1561 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1621 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     1681 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     1741 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     1801 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1861 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     1921 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     1981 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2041 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2101 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2161 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2221 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2281 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2341 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2401 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2461 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2521 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2581 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     2641 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     2701 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     2761 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     2821 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     2881 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     2941 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3001 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3061 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3121 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     3181 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     3241 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     3301 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     3361 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     3421 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     3481 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc aacgctgccc
     3541 gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctgccaaggg
     3601 ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct tggagtggtg
     3661 aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac
     3721 cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gcctacaatc catgccaacc
     3781 cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg
     3841 aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta
     3901 cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa
     3961 tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg
     4021 cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     4081 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     4141 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     4201 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     4261 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     4321 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4381 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4441 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4501 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4561 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4621 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     4681 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     4741 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     4801 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     4861 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     4921 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     4981 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5041 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5101 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5161 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5221 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5281 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5341 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5401 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5461 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     5521 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     5581 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     5641 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     5701 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     5761 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     5821 gatcccgcga aat
//
