LOCUS       sequence-436823-        6908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of AcK in E. coli BL21_DE3 (pLys) or similar.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6908)
  TITLE     Efficient synthetic access to stable isotope labelled pseudouridine 
            phosphoramidites for RNA NMR spectroscopy
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6908)
  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            "Unpublished"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: pet15b_ack
FEATURES             Location/Qualifiers
     source          1..6908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(96..1178)
                     /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     1140..1159
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1179..1256)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1462..1481
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1565..1584
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1565..1583
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1584..1608
                     /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             1623..1645
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1664..1681
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1691..1708
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2970..2988)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2984..3031
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3206..3211,3212..3228,3229..3234))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3261..3279)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3347..3451
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3452..3520,3521..4312)
                     /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(3670..3689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4483..5071
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4972..4991
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5225..5242
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5257..5399
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5325..5344)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5485..5507
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5501..5692)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
       61 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      121 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
      181 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
      241 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
      301 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
      361 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
      421 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
      481 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
      541 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
      601 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
      661 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
      721 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
      781 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
      841 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
      901 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
      961 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     1021 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     1081 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     1141 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1201 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     1261 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     1321 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     1381 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     1441 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     1501 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     1561 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     1621 attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca
     1681 cagcagcggc ctggtgccgc gcggcagcca tatgtcgagt aagttagtac tggttctgaa
     1741 ctgcggtagt tcttcactga aatttgccat catcgatgca gtaaatggtg aagagtacct
     1801 ttctggttta gccgaatgtt tccacctgcc cgaagcacgt atcaaatgga aaatggacgg
     1861 caataaacag gaagcggctt taggtgcagg cgccgctcac agcgaagcgc tcaactttat
     1921 cgttaatact attctggcac aaaaaccaga actgtctgcg cagctgactg ctatcggtca
     1981 ccgtatcgta cacggcggcg aaaagtatac cagctccgta gtgatcgatg agtctgttat
     2041 tcagggtatc aaagatgcag cttcttttgc accgctgcac aacccggctc acctgatcgg
     2101 tatcgaagaa gctctgaaat ctttcccaca gctgaaagac aaaaacgttg ctgtatttga
     2161 caccgcgttc caccagacta tgccggaaga gtcttacctc tacgccctgc cttacaacct
     2221 gtacaaagag cacggcatcc gtcgttacgg cgcgcacggc accagccact tctatgtaac
     2281 ccaggaagcg gcaaaaatgc tgaacaaacc ggtagaagaa ctgaacatca tcacctgcca
     2341 cctgggcaac ggtggttccg tttctgctat ccgcaacggt aaatgcgttg acacctctat
     2401 gggcctgacc ccgctggaag gtctggtcat gggtacccgt tctggtgata tcgatccggc
     2461 gatcatcttc cacctgcacg acaccctggg catgagcgtt gacgcaatca acaaactgct
     2521 gaccaaagag tctggcctgc tgggtctgac cgaagtgacc agcgactgcc gctatgttga
     2581 agacaactac gcgacgaaag aagacgcgaa gcgcgcaatg gacgtttact gccaccgcct
     2641 ggcgaaatac atcggtgcct acactgcgct gatggatggt cgtctggacg ctgttgtatt
     2701 cactggtggt atcggtgaaa atgccgcaat ggttcgtgaa ctgtctctgg gcaaactggg
     2761 cgtgctgggc tttgaagttg atcatgaacg caacctggct gcacgtttcg gcaaatctgg
     2821 tttcatcaac aaagaaggta cccgtcctgc ggtggttatc ccaaccaacg aagaactggt
     2881 tatcgcgcaa gacgcgagcc gcctgactgc ctgactcgag gatccggctg ctaacaaagc
     2941 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     3001 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc
     3061 cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg
     3121 gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag
     3181 caatttaact gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga
     3241 gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3301 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3361 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3421 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3481 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3541 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3601 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3661 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     3721 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3781 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3841 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3901 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3961 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     4021 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4081 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4141 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4201 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4261 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4321 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4381 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4441 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4501 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4561 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4621 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4681 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4741 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4801 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4861 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4921 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4981 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5041 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5101 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5161 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5221 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     5281 gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat ctgctctgat
     5341 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     5401 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5461 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5521 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     5581 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     5641 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     5701 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     5761 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     5821 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     5881 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     5941 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     6001 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     6061 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     6121 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg
     6181 agatgcgccg cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt
     6241 tggtttgcgc attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga
     6301 atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc
     6361 gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc
     6421 gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc cagtgatcga
     6481 agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac
     6541 ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat
     6601 cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc
     6661 gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     6721 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     6781 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     6841 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     6901 ccgcgccc
//
