LOCUS       sequence-417773-        3980 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses inactive 2',3'-cyclic nucleotide phosphodiesterase 
            catalytic domain (catalytic residues mutated).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3980)
  AUTHORS   Fontaine BM, Martin KS, Garcia-Rodriguez JM, Jung C, Briggs L, 
            Southwell JE, Jia X, Weinert EE
  TITLE     RNase I regulates Escherichia coli 2',3'-cyclic nucleotide 
            monophosphate levels and biofilm formation.
  JOURNAL   Biochem J. 2018 Apr 30;475(8):1491-1506. doi: 10.1042/BCJ20170906.
  PUBMED    29555843
REFERENCE   2  (bases 1 to 3980)
  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"; journalName: 
            "Biochem J"; date: "2018-04-30- 30"; volume: "475"; issue: "8"; 
            pages: "1491-1506"
COMMENT     Sequence Label: pKT-CNP-inact
FEATURES             Location/Qualifiers
     source          1..3980
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      69..657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     558..577
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(719..1345)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1250..1269
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        1361..1416
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1367..1385
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    1397..1415
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        1440..1458
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1490..1512
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        complement(2249..2267)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2274..2291)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2274..2290)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2283..2305)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2432..2887
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2519..2538)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2729..2750
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2913..3017
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3018..3086,3087..3878)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3236..3255)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
       61 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
      121 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
      181 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
      241 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
      301 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
      361 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
      421 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
      481 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
      541 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
      601 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
      661 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgctt
      721 aagacccact ttcacattta agttgttttt ctaatccgca aatgatcaat tcaaggccga
      781 ataagaaggc tggctctgca ccttggtgat caaataattc gatagcttgt cgtaataatg
      841 gcggcatact atcagtagta ggtgtttccc tttcttcttt agcgacttga tgctcttgat
      901 cttccaatac gcaacctaaa gtaaaatgcc ccacagcgct gagtgcatat aatgcattct
      961 ctagtgaaaa accttgttgg cataaaaagg ctaattgatt ttcgagagtt tcatactgtt
     1021 tttctgtagg ccgtgtacct aaatgtactt ttgctccatc gcgatgactt agtaaagcac
     1081 atctaaaact tttagcgtta ttacgtaaaa aatcttgcca gctttcccct tctaaagggc
     1141 aaaagtgagt atggtgccta tctaacatct caatggctaa ggcgtcgagc aaagcccgct
     1201 tattttttac atgccaatac aatgtaggct gctctacacc tagcttctgg gcgagtttac
     1261 gggttgttaa accttcgatt ccgacctcat taagcagctc taatgcgctg ttaatcactt
     1321 tacttttatc taatctcgac atcattaatt cctaattttt gttgacactc tatcattgat
     1381 agagttattt taccactccc tatcagtgat agagaaaagt ctagccgatc ccgcgaaatt
     1441 aatacgactc actataggga gaccacaacg gtttccctct agaaataatt ttgtttaact
     1501 ttaagaagga gatatacata tgggcctgga aaaagatttc ctgccgctgt actttggctg
     1561 gttcctgacg aaaaaatcga gcgaaaccct gcgtaaagct ggtcaagtgt tcctggaaga
     1621 actgggcaac cataaagcgt ttaagaaaga actgcgtcac ttcatttctg gtgatgaacc
     1681 gaaagaaaaa ctggacctgg tcagctattt tggcaaacgc ccgccgggtg ttctgctgtg
     1741 caccacgaaa ttctgtgatt atggcaaagc gaccggtgcc gaagaatacg cccagcaaga
     1801 tgtggttcgt cgctcttacg gcaaagcatt taaactgtcc atctcagctc tgttcgtgac
     1861 cccgaaaacg gcaggtgctc aggtcgtgct gaatgaacag gaactgcaac tgtggccgag
     1921 cgatctggac aaaccgagct ctagtgaaag cctgccgccg ggcagccgtg cgctggttac
     1981 cctgggttgt gccgcggatg ttcagccggt ccaaacgggc ctggacctgc tggaaattct
     2041 gcagcaagtg aaaggcggtt cccagggcga agaagttggt gaactgccgc gtggtaaact
     2101 gtatagcctg ggcaaaggtc gctggatgct gtccctggca aagaaaatgg aagtgaaagc
     2161 tatctttacg ggctactacg gctgaactag tgcggccgct tgataaagcc cgcctaatga
     2221 gcgggctttt ttttgagctc caattcgccc tatagtgagt cgtattacaa ttcactggcc
     2281 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     2341 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     2401 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     2461 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2521 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2581 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2641 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2701 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2761 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2821 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     2881 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2941 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3001 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3061 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3121 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3181 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3241 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     3301 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3361 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3421 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3481 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3541 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3601 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3661 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3721 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3781 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3841 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3901 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3961 tttttgataa tctcatgacc
//
