LOCUS       sequence-436827-        6632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Rbsk in E. coli BL21_DE3 (pLys) or similar.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6632)
  TITLE     Efficient synthetic access to stable isotope labelled pseudouridine 
            phosphoramidites for RNA NMR spectroscopy
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6632)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pet15_rbsk
FEATURES             Location/Qualifiers
     source          1..6632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     109..128
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(347..451)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     519..537
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(564..569,570..586,587..592)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(767..814)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     810..828
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1817..1834)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1844..1861)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1880..1902
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1917..1941
                     /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(1941..1960)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1942..1960)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2044..2063)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2269..2346
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2347..3429
                     /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(2366..2385)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4741..4932
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4926..4948)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5034..5173
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5089..5108
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5188..5205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5359..5947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5439..5458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6118..6632,1..277,278..346))
                     /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"
ORIGIN
        1 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
       61 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
      121 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
      181 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
      241 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
      301 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
      361 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
      421 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
      481 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
      541 ttcgtcttca agaattctca tgtttgacag cttatcatcg ataagcttta atgcggtagt
      601 ttatcacagt taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc
      661 atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta
      721 ctgccgggcc tcttgcggga tatccggata tagttcctcc tttcagcaaa aaacccctca
      781 agacccgttt agaggcccca aggggttatg ctagttattg ctcagcggtg gcagcagcca
      841 actcagcttc ctttcgggct ttgttagcag ccggatcctc gagtcacctc tgcctgtcta
      901 aaaatgcgtc gatctcttca cgccacggta cggaaggttg tgcgccttta cgtgttacgg
      961 caatcgcagc ggcagcatgg gcaaaacgaa tcgcctctgg caatggtttt tcttccagca
     1021 atgccgtgat taacgcaccg ttaaaggtat ctccggcagc aatggtatcg acagcctgca
     1081 cccggaatcc aggaacgcgc tgaccttcac cattcacgct agcccataca ccacgacttc
     1141 ctaaagtaat cagtacagta cggatacctt tttcatgcag tacctgcgcc gccttcgctg
     1201 catcttcatc attttcaaca cgaataccgg tgagcttttc tgcttccgtt tcgtttggcg
     1261 taataatgtc caccagcgcc agcagttcgt caggaagttc gcgagccgga gccgggttaa
     1321 gcgcaacgat agtcttattt tgatgggcga ttttcgccgc tgccatcaca ctttcgagtg
     1381 gtgattccag ctgcattaat aatgctgacg cgttggcaat acgctcacgt tgcgcttcca
     1441 ccagcgccgg ggaaagggca gcattagcgc cggcatgaat accgatgaca ttctcacctt
     1501 cgccattaac aaaaatcagc gccacacctg ttgattcgcc tttgatcacg ctgaccggag
     1561 taatatcaat gttatcagtg gcgagctgct ggcgaacgct ctcaccaatg ctgtcatcac
     1621 ccgtacaggc aataaacgcg atattcgcac cgctacgccc agcagccaca gcctgattcg
     1681 cgcctttgcc gccaaatgca acctgatagt ggttaccggt tacggtttcg cctggagtag
     1741 gaaaagattg aagattaaga atgtggtcag cattaatgct gccaagaaca acgaggctgc
     1801 ctgcgttttg catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat
     1861 ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa
     1921 ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct
     1981 cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
     2041 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
     2101 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
     2161 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
     2221 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa
     2281 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
     2341 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
     2401 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     2461 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     2521 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     2581 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     2641 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     2701 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     2761 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     2821 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     2881 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     2941 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     3001 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     3061 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     3121 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     3181 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     3241 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     3301 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     3361 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     3421 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg
     3481 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
     3541 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
     3601 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct
     3661 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc
     3721 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg acgcgctggg
     3781 ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta tgattcttct
     3841 cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga
     3901 cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa cttcgatcat
     3961 tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga acgggttggc
     4021 atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg
     4081 gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact
     4141 ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttgg
     4201 cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat ctcgggcagc
     4261 gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc
     4321 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc
     4381 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt
     4441 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc
     4501 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg
     4561 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc
     4621 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc
     4681 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca
     4741 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
     4801 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
     4861 cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt
     4921 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     4981 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     5041 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     5101 agattgtact gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa
     5161 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5221 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5281 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5341 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5401 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5461 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5521 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5581 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5641 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5701 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5761 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     5821 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5881 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5941 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6001 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6061 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6121 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6181 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6241 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6301 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6361 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6421 tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6481 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6541 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6601 ggttatggca gcactgcata attctcttac tg
//
