LOCUS       sequence-433566-        5479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET31b plasmid with short insert lacking ribosome binding site to 
            increase 2',3'-cNMP levels.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5479)
  AUTHORS   Chauhan SS, Marotta NJ, Karls AC, Weinert EE
  TITLE     Binding of 2',3'-Cyclic Nucleotide Monophosphates to Bacterial 
            Ribosomes Inhibits Translation.
  JOURNAL   ACS Cent Sci. 2022 Nov 23;8(11):1518-1526. doi: 
            10.1021/acscentsci.2c00681. Epub 2022 Sep 21.
  PUBMED    36439312
REFERENCE   2  (bases 1 to 5479)
  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"; doi: 
            "10.1021/acscentsci.2c00681"; journalName: "ACS Cent Sci"; date: 
            "2022-11-23- 23"; volume: "8"; issue: "11"; pages: "1518-1526"
COMMENT     Sequence Label: pNoRBS
FEATURES             Location/Qualifiers
     source          1..5479
                     /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"
     terminator      complement(164..211)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(282..300)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(370..389)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        595..672
                     /gene="lacI"
                     /label=lacI promoter
     CDS             673..1755
                     /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(692..711)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2564..2755
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2749..2771)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2857..2996
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2912..2931
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3011..3028)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3262..3281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4022..4813,4814..4882))
                     /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     4645..4664
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4883..4987)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5013..5468)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5150..5171)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5362..5381
                     /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 gagcaaaaaa cccctcaaga
      181 cccgtttaga ggccccaagg ggttatgcta gggctgtacg agggcgctca cactctactc
      241 aacagcgcga acgctggacc cgtccttcgc cgacgactag tcctatagtg agtcgtatta
      301 agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
      361 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
      421 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
      481 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
      541 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
      601 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
      661 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
      721 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
      781 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
      841 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
      901 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
      961 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     1021 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     1081 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     1141 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     1201 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     1261 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     1321 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     1381 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     1441 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     1501 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     1561 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     1621 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     1681 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     1741 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     1801 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     1861 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     1921 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     1981 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     2041 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
     2101 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     2161 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     2221 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     2281 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     2341 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     2401 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     2461 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     2521 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     2581 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     2641 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     2701 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     2761 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     2821 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     2881 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     2941 caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     3001 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3061 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagcacatc
     3541 ctgtaaccag ctctggtgca tggcctgcac gtaacctttt accgtttcag cacggctcgg
     3601 catcgctccg tcgatccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3661 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     3721 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3781 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3841 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3901 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3961 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4021 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4081 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4141 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4201 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4261 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4321 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4381 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4441 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4501 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4561 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4621 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4681 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4741 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4801 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4861 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4921 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4981 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     5041 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     5101 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     5161 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     5221 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     5281 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     5341 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     5401 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5461 gggcgcgtcc cattcgcca
//
