LOCUS       sequence-388086-        4729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative selection plasmid for selecting ncAA-encoding 
            Methanomethylophilus alvus Pyl-RS mutants. Expresses 2xTAG-codon 
            interrupted barnase gene and M. alvus Pyl-tRNA(6). p15a origin of 
            replication..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4729)
  AUTHORS   Avila-Crump S, Hemshorn ML, Jones CM, Mbengi L, Meyer K, Griffis JA,
            Jana S, Petrina GE, Pagar VV, Karplus PA, Petersson EJ, Perona JJ, 
            Mehl RA, Cooley RB
  TITLE     Generating Efficient Methanomethylophilus alvus Pyrrolysyl-tRNA 
            Synthetases for Structurally Diverse Non-Canonical Amino Acids.
  JOURNAL   ACS Chem Biol. 2022 Dec 16;17(12):3458-3469. doi: 
            10.1021/acschembio.2c00639. Epub 2022 Nov 16.
  PUBMED    36383641
REFERENCE   2  (bases 1 to 4729)
  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/acschembio.2c00639"; journalName: "ACS Chem Biol"; date: 
            "2022-12-16- 16"; volume: "17"; issue: "12"; pages: "3458-3469"
COMMENT     Sequence Label: pNeg-Ma-barnase-TAG3-TAG45
FEATURES             Location/Qualifiers
     source          1..4729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(185..1063)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1090..1374
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1297..1316
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1408..1743
                     /codon_start=1
                     /product="ribonuclease from Bacillus amyloliquefaciens"
                     /label=barnase
                     /note="The barnase gene is lethal in standard bacterial 
                     transformation strains."
                     /translation="MG*VINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLA*
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     primer_bind     complement(1817..1834)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1987..2073
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2165..2192
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(2423..3082)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFETFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(3083..3185)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3713..3729)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3722..3744)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3920..3937)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4054..4599)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4134..4153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccggaaggag ctgactgggt tgaaggctct catcaccgtc atcaccgaaa cgcgcgaggc
       61 agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcata atgtgcctgt caaatggacg
      121 aagcagggat tctgcaaacc ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta
      181 ccaattatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
      241 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
      301 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
      361 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
      421 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
      481 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
      541 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
      601 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
      661 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
      721 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
      781 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
      841 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
      901 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
      961 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
     1021 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccgc
     1081 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
     1141 actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
     1201 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
     1261 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
     1321 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
     1381 tttttgggct aacaggagga attaaccatg ggataggtta tcaacacgtt tgacggggtt
     1441 gcggattatc ttcagacata tcataagcta cctgataatt acattacaaa atcagaagca
     1501 caagccctcg gctgggtggc atcaaaaggg aaccttgcat aggtcgctcc ggggaaaagc
     1561 atcggcggag acatcttctc aaacagggaa ggcaaactcc cgggcaaaag cggacgaaca
     1621 tggcgtgaag cggatattaa ctatacatca ggcttcagaa attcagaccg gattctttac
     1681 tcaagcgact ggctgattta caaaacaacg gaccattatc agacctttac aaaaatcaga
     1741 taactcgaga tctgcagctg gtaccatatg ggaattcgaa gcttggctgt tttggcggat
     1801 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     1861 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     1921 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     1981 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     2041 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     2101 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     2161 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tcaggcattt
     2221 gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca
     2281 taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat
     2341 ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc gtttaagggc
     2401 accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta
     2461 attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg
     2521 ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatagtg aaaacggggg
     2581 cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat
     2641 tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac
     2701 cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt
     2761 cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa
     2821 cactatccca tatcaccagc tcaccgtctt tcattgccat acggaactcc ggatgagcat
     2881 tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta
     2941 cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa
     3001 ctgactgaaa tgtctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat
     3061 atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa
     3121 aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc
     3181 gatcaacgtc tcattttcgc caaaagttgg cccagggctt cccggtatca acagggacac
     3241 caggatttat ttattctgcg aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg
     3301 cgtcgggtga tgctgccaac ttactgattt agtgtatgat ggtgtttttg aggtgctcca
     3361 gtggcttctg tttctatcag ctgtccctcc tgttcagcta ctgacggggt ggtgcgtaac
     3421 ggcaaaagca ccgccggaca tcagcgctag cggcgttctg ttgcccgtct cactggtgaa
     3481 aagaaaaaca accctggcgc cgcttctttg agcgaacgat caaaaataag tggcgcccca
     3541 tcaaaaaaat attctcaaca taaaaaactt tgtgtaatac ttgtaacgct agatctgggg
     3601 gacggtccgg cgaccagcgg gtctctaaaa cctagcatag cggggttcga caccccggtc
     3661 tctcgccaac tagtatcctt agcgaaagct aaggattttt tttaagcttg gcactggccg
     3721 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     3781 cacatccccc tttcgccagg tatactggct tactatgttg gcactgatga gggtgtcagt
     3841 gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat
     3901 acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc
     3961 gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta
     4021 acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa
     4081 gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata
     4141 ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac
     4201 cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg
     4261 ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct
     4321 gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac
     4381 tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa
     4441 ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc
     4501 aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt
     4561 tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag
     4621 ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     4681 cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgccca
//
