LOCUS       sequence-444187-        5850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescence control plasmid for selecting ncAA-encoding 
            Methanomethylophilus alvus Pyl-RS mutants. Expresses SUMO-sfGFP-WT 
            and M. alvus Pyl-tRNA(6). p15a origin. Tetracycline resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5850)
  TITLE     Engineering of Aminoacyl-tRNA synthetase/tRNA pairs for efficient 
            genetic encoding of non-canonical amino acids into proteins.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5850)
  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            "Unpublished"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: pALS3-Ma-SUMO-sfGFP-WT
FEATURES             Location/Qualifiers
     source          1..5850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      18..563
                     /direction=RIGHT
                     /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     464..483
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     680..697
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(878..2068)
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     1697..1716
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(join(2116..2121,2122..2138,2139..2144))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     2276..2298
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2291..2307
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(2864..2891)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2983..3069)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3222..3239
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(3308..3325)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(3332..4042,4043..4045))
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             complement(4061..4357)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTP
                     LRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     promoter        complement(4391..4675)
                     /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     complement(4449..4468)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4702..5580
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac
       61 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac
      121 cgaggtaact ggcttggagg agcggagtca ccaaaacttg tcctttcagt ttagccttaa
      181 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg
      241 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      301 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga
      361 actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa
      421 accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaaac gcctggtatc
      481 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt
      541 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag
      601 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg taagccattt ccgctcgccg
      661 cagtcgaacg accgagcgta gcgagtcagt gagcgaggaa gcgggaatgg tgcatgcaag
      721 gagcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc
      781 caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga
      841 gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc
      901 atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg
      961 ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa
     1021 tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt
     1081 catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga
     1141 gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc
     1201 ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     1261 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     1321 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     1381 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     1441 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     1501 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     1561 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     1621 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     1681 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     1741 cggtgatgcc ggccacgatg cgtccggcgt agaggatcca caggacgggt gtggtcgcca
     1801 tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg cggcggccaa
     1861 agcggtcgga cagtgctccg agaacgggtg cgcatagaaa ttgcatcaac gcatatagcg
     1921 ctagcagcac gccatagtga ctggcgatgc tgtcggaatg gacgatatcc cgcaagaggc
     1981 ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga
     2041 tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact
     2101 gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga gaattacaac
     2161 ttatatcgta tggggctgac ttcaggtgct acattgcagg agtcgcataa gggagagcgt
     2221 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     2281 tcacgacgtt gtaaaacgac ggccagtgcc aagcttaaaa aaaatcctta gctttcgcta
     2341 aggatactag ttggcgagag accggggtgt cgaaccccgc tatgctaggt tttagagacc
     2401 cgctggtcgc cggaccgtcc cccagatcta gcgttacaag tattacacaa agttttttat
     2461 gttgagaata tttttttgat ggggcgccac ttatttttga tcgttcgctc aaagaagcgg
     2521 cgccagggtt gtttttcttt tcaccagtga gacgggcaac agaacgccat gagcggcctc
     2581 atttcttatt ctgagttaca acagtccgca ccgctgccgg tagctccttc cggtgggcgc
     2641 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     2701 aggtgccggc agcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     2761 aacaagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     2821 gtggaacacc tacatctgtg tcgacagagt ttgtagaaac gcaaaaaggc catccgtcag
     2881 gatggccttc tgcttaattt gatgcctggc agtttatggc gggcgtcctg cccgccaccc
     2941 tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag
     3001 agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg
     3061 ttttatttga tgcctggcag ttccctactc tcgcatgggg agaccccaca ctaccatcgg
     3121 cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc
     3181 cgccaggcaa attctgtttt atcagaccgc ttctgcgttc tgatttaatc tgtatcaggc
     3241 tgaaaatctt ctctcatccg ccaaaacagc caagcttcga attcccatat ggtaccagct
     3301 gcagttaatg gtgatgatga tggtggctgc ctttatacag ttcatccata ccgtgggtaa
     3361 tgcccgcggc ggtaacaaat tccagcagca ccatatgatc acgtttttca ttcggatctt
     3421 tgctcagaac gctctgggtg ctcagataat gattatccgg cagcagcacc gggccatcac
     3481 caatcggggt attctgctga taatgatccg ccagctgcac gctaccatct tccacgttgt
     3541 gacggatttt aaagttcgct ttgatgccat ttttctgttt atcggcggta atatacacat
     3601 tatggctgtt gaaattatat tccagtttat gacccagaat gttgccatct tctttaaaat
     3661 caataccttt cagttcaatg cggttcacca gggtatcgcc ttcaaattta acttccgcac
     3721 gggttttata ggtgccatca tctttgaagc taatggtacg ttcctgcaca tagccttccg
     3781 gcatcgcgct tttaaagaaa tcatggcgtt tcatatgatc cggatagcgg ctaaagcact
     3841 gaacgccata ggtcagggtg gtcaccaggg tcggccacgg aaccggcagt ttaccggtgg
     3901 tgcaaataaa tttcagggtc agtttaccgt tggtcgcatc accttcgcct tcgccacgaa
     3961 cgctaaattt atggccattc acatcaccat ccagttccac cagaatcggc acaacgccgg
     4021 taaacagttc ttcacctttg ctaacagagc cggtaccgga tccaccaatc tgttctctgt
     4081 gagcctcaat aatatcgtta tcctccatgt ccaaatcttc aggggtctga tcagcttgga
     4141 ttctaatacc gtcgtacaag aatcttaagg agtccatttc cttaccctgt cttttagcga
     4201 acgcttccat cagccttctt aaaggagtgg tctttttgat cttgaagaag atctctgaag
     4261 atccatcgga cacctttaaa ttgatgtgag tctcaggctt gacttctggc ttgacctctg
     4321 gcttagcttc ttgattgact tctgagtcgc tgcccatggt taattcctcc tgttagccca
     4381 aaaaacgggt atggagaaac agtagagagt tgcgataaaa agcgtcaggt aggatccgct
     4441 aatcttatgg ataaaaatgc tatggcatag caaagtgtga cgccgtgcaa ataatcaatg
     4501 tggacttttc tgccgtgatt atagacactt ttgttacgcg tttttgtcat ggctttggtc
     4561 ccgctttgtt acagaatgct tttaataagc ggggttaccg gtttggttag cgagaagagc
     4621 cagtaaaaga cgcagtgacg gcaatgtctg atgcaatatg gacaattggt ttcttctctg
     4681 aatggcggga gtatgaaaag tatggctgaa gcgcaaaatg atcccctgct gccgggatac
     4741 tcgtttaatg cccatctggt ggcgggttta acgccgattg aggccaacgg ttatctcgat
     4801 ttttttatcg accgaccgct gggaatgaaa ggttatattc tcaatctcac cattcgcggt
     4861 cagggggtgg tgaaaaatca gggacgagaa tttgtttgcc gaccgggtga tattttgctg
     4921 ttcccgccag gagagattca tcactacggt cgtcatccgg aggctcgcga atggtatcac
     4981 cagtgggttt actttcgtcc gcgcgcctac tggcatgaat ggcttaactg gccgtcaata
     5041 tttgccaata cggggttctt tcgcccggat gaagcgcacc agccgcattt cagcgacctg
     5101 tttgggcaaa tcattaacgc cgggcaaggg gaagggcgct attcggagct gctggcgata
     5161 aatctgcttg agcaattgtt actgcggcgc atggaagcga ttaacgagtc gctccatcca
     5221 ccgatggata atcgggtacg cgaggcttgt cagtacatca gcgatcacct ggcagacagc
     5281 aattttgata tcgccagcgt cgcacagcat gtttgcttgt cgccgtcgcg tctgtcacat
     5341 cttttccgcc agcagttagg gattagcgtc ttaagctggc gcgaggacca acgtatcagc
     5401 caggcgaagc tgcttttgag caccacccgg atgcctatcg ccaccgtcgg tcgcaatgtt
     5461 ggttttgacg atcaactcta tttctcgcgg gtatttaaaa aatgcaccgg ggccagcccg
     5521 agcgagttcc gtgccggttg tgaagaaaaa gtgaatgatg tagccgtcaa gttgtcataa
     5581 ttggtaacga atcagacaat tgacggcttg acggagtagc atagggtttg cagaatccct
     5641 gcttcgtcca tttgacaggc acattatgca tgccgcttcg ccttcgcgcg cgaattgatc
     5701 tgctgcctcg cgcgtttcgg tgatgacggt gagatcctgg gcgcggggca tgactaacat
     5761 gagaattaca acttatatcg tatggggctg acttcaggtg ctacatttga agagataaat
     5821 tgcactgaaa tctagaaata ttttatctga
//
