LOCUS       sequence-444184-        5832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescence plasmid for selecting ncAA-encoding 
            Methanomethylophilus alvus Pyl-RS mutants. Expresses 
            SUMO-TAG35-sfGFP 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 5832)
  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  JOURNAL   Unpublished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 5832)
  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            "Unpublished"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-TAG35-sfGFP
FEATURES             Location/Qualifiers
     source          1..5832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(265..270,271..287,288..293))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     426..448
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     441..457
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(1014..1041)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1133..1219)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1372..1389
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(1458..1475)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(1482..2192,2193..2195))
                     /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(2211..2507)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSS*IFFKIKKTTP
                     LRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     promoter        complement(2541..2825)
                     /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(2599..2618)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             2852..3730
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     rep_origin      4018..4563
                     /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     4464..4483
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4680..4697
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(join(4859..5832,1..217))
                     /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     5678..5697
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggcggccaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg
       61 catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc
      121 gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg
      181 tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc
      241 aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag
      301 aattacaact tatatcgtat ggggctgact tcaggtgcta catttgcagg agtcgcataa
      361 gggagagcgt ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      421 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagcttaaaa aaaatcctta
      481 gctttcgcta aggatactag ttggcgagag accggggtgt cgaaccccgc tatgctaggt
      541 tttagagacc cgctggtcgc cggaccgtcc cccagatcta gcgttacaag tattacacaa
      601 agttttttat gttgagaata tttttttgat ggggcgccac ttatttttga tcgttcgctc
      661 aaagaagcgg cgccagggtt gtttttcttt tcaccagtga gacgggcaac agaacgccat
      721 gagcggcctc atttcttatt ctgagttaca acagtccgca ccgctgccgg tagctccttc
      781 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
      841 ctcgtaggac aggtgccggc agcgcccaac agtcccccgg ccacggggcc tgccaccata
      901 cccacgccga aacaagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
      961 tggctaccct gtggaacacc tacatctgtg tcgacagagt ttgtagaaac gcaaaaaggc
     1021 catccgtcag gatggccttc tgcttaattt gatgcctggc agtttatggc gggcgtcctg
     1081 cccgccaccc tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc
     1141 tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact
     1201 gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcatgggg agaccccaca
     1261 ctaccatcgg cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc
     1321 gcgctactgc cgccaggcaa attctgtttt atcagaccgc ttctgcgttc tgatttaatc
     1381 tgtatcaggc tgaaaatctt ctctcatccg ccaaaacagc caagcttcga attcccatat
     1441 ggtaccagct gcagttaatg gtgatgatga tggtggctgc ctttatacag ttcatccata
     1501 ccgtgggtaa tgcccgcggc ggtaacaaat tccagcagca ccatatgatc acgtttttca
     1561 ttcggatctt tgctcagaac gctctgggtg ctcagataat gattatccgg cagcagcacc
     1621 gggccatcac caatcggggt attctgctga taatgatccg ccagctgcac gctaccatct
     1681 tccacgttgt gacggatttt aaagttcgct ttgatgccat ttttctgttt atcggcggta
     1741 atatacacat tatggctgtt gaaattatat tccagtttat gacccagaat gttgccatct
     1801 tctttaaaat caataccttt cagttcaatg cggttcacca gggtatcgcc ttcaaattta
     1861 acttccgcac gggttttata ggtgccatca tctttgaagc taatggtacg ttcctgcaca
     1921 tagccttccg gcatcgcgct tttaaagaaa tcatggcgtt tcatatgatc cggatagcgg
     1981 ctaaagcact gaacgccata ggtcagggtg gtcaccaggg tcggccacgg aaccggcagt
     2041 ttaccggtgg tgcaaataaa tttcagggtc agtttaccgt tggtcgcatc accttcgcct
     2101 tcgccacgaa cgctaaattt atggccattc acatcaccat ccagttccac cagaatcggc
     2161 acaacgccgg taaacagttc ttcacctttg ctaacagagc cggtaccgga tccaccaatc
     2221 tgttctctgt gagcctcaat aatatcgtta tcctccatgt ccaaatcttc aggggtctga
     2281 tcagcttgga ttctaatacc gtcgtacaag aatcttaagg agtccatttc cttaccctgt
     2341 cttttagcga acgcttccat cagccttctt aaaggagtgg tctttttgat cttgaagaag
     2401 atctatgaag atccatcgga cacctttaaa ttgatgtgag tctcaggctt gacttctggc
     2461 ttgacctctg gcttagcttc ttgattgact tctgagtcgc tgcccatggt taattcctcc
     2521 tgttagccca aaaaacgggt atggagaaac agtagagagt tgcgataaaa agcgtcaggt
     2581 aggatccgct aatcttatgg ataaaaatgc tatggcatag caaagtgtga cgccgtgcaa
     2641 ataatcaatg tggacttttc tgccgtgatt atagacactt ttgttacgcg tttttgtcat
     2701 ggctttggtc ccgctttgtt acagaatgct tttaataagc ggggttaccg gtttggttag
     2761 cgagaagagc cagtaaaaga cgcagtgacg gcaatgtctg atgcaatatg gacaattggt
     2821 ttcttctctg aatggcggga gtatgaaaag tatggctgaa gcgcaaaatg atcccctgct
     2881 gccgggatac tcgtttaatg cccatctggt ggcgggttta acgccgattg aggccaacgg
     2941 ttatctcgat ttttttatcg accgaccgct gggaatgaaa ggttatattc tcaatctcac
     3001 cattcgcggt cagggggtgg tgaaaaatca gggacgagaa tttgtttgcc gaccgggtga
     3061 tattttgctg ttcccgccag gagagattca tcactacggt cgtcatccgg aggctcgcga
     3121 atggtatcac cagtgggttt actttcgtcc gcgcgcctac tggcatgaat ggcttaactg
     3181 gccgtcaata tttgccaata cggggttctt tcgcccggat gaagcgcacc agccgcattt
     3241 cagcgacctg tttgggcaaa tcattaacgc cgggcaaggg gaagggcgct attcggagct
     3301 gctggcgata aatctgcttg agcaattgtt actgcggcgc atggaagcga ttaacgagtc
     3361 gctccatcca ccgatggata atcgggtacg cgaggcttgt cagtacatca gcgatcacct
     3421 ggcagacagc aattttgata tcgccagcgt cgcacagcat gtttgcttgt cgccgtcgcg
     3481 tctgtcacat cttttccgcc agcagttagg gattagcgtc ttaagctggc gcgaggacca
     3541 acgtatcagc caggcgaagc tgcttttgag caccacccgg atgcctatcg ccaccgtcgg
     3601 tcgcaatgtt ggttttgacg atcaactcta tttctcgcgg gtatttaaaa aatgcaccgg
     3661 ggccagcccg agcgagttcc gtgccggttg tgaagaaaaa gtgaatgatg tagccgtcaa
     3721 gttgtcataa ttggtaacga atcagacaat tgacggcttg acggagtagc atagggtttg
     3781 cagaatccct gcttcgtcca tttgacaggc acattatgca tgccgcttcg ccttcgcgcg
     3841 cgaattgatc tgctgcctcg cgcgtttcgg tgatgacggt gagatcctgg gcgcggggca
     3901 tgactaacat gagaattaca acttatatcg tatggggctg acttcaggtg ctacatttga
     3961 agagataaat tgcactgaaa tctagaaata ttttatctga ttaataagat gatcttcttg
     4021 agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg
     4081 cggtttttcg aaggttctct gagctaccaa ctctttgaac cgaggtaact ggcttggagg
     4141 agcgaagtca ccaaaacttg tcctttcagt ttagccttaa ccggcgcatg acttcaagac
     4201 taactcctct aaatcaatta ccagtggctg ctgccagtgg tgcttttgca tgtctttccg
     4261 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcggactga acggggggtt
     4321 cgtgcataca gtccagcttg gagcgaactg cctacccgga actgagtgtc aggcgtggaa
     4381 tgagacaaac gcggccataa cagcggaatg acaccggtaa accgaaaggc aggaacagga
     4441 gagcgcacga gggagccgcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4501 cgccaccact gatttgagcg tcagatttcg tgatgcttgt caggggggcg gagcctatgg
     4561 aaaaacggct ttgccgcggc cctctcactt ccctgttaag tatcttcctg gcatcttcca
     4621 ggaaatctcc gccccgttcg taagccattt ccgctcgccg cagtcgaacg accgagcgta
     4681 gcgagtcagt gagcgaggaa gcggcccgag atgcgccgcg tgcggctgct ggagatggcg
     4741 gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat
     4801 tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg gcttccattc
     4861 aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata
     4921 gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg
     4981 ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt
     5041 gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca
     5101 tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg
     5161 cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct
     5221 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     5281 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     5341 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     5401 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     5461 aggctctcaa gggcatcggt cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     5521 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     5581 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     5641 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5701 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcc
     5761 acaggacggg tgtggtcgcc atgatcgcgt agtcgatagt ggctccaagt agcgaagcga
     5821 gcaggactgg gc
//
