LOCUS       sequence-444185-        5551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescence control plasmid for selecting ncAA-encoding 
            Methanomethylophilus alvus Pyl-RS mutants. Expresses 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 5551)
  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  JOURNAL   Unpublished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 5551)
  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            "Unpublished"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-sfGFP-WT
FEATURES             Location/Qualifiers
     source          1..5551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(997..1014)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1131..1676)
                     /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(1211..1230)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1964..2842)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2869..3153
                     /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     3076..3095
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             join(3187..3189,3190..3192,3193..3903)
                     /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="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             3910..3927
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4006..4023)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4176..4262
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4354..4381
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4938..4954)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4947..4969)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(5101..5106,5107..5123,5124..5129)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             join(5177..5551,1..816)
                     /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     complement(5529..5548)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
       61 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
      121 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
      181 aagggagagc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
      241 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
      301 ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg
      361 acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc
      421 gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg
      481 gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc
      541 attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc
      601 aggcaggtag atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc
      661 ctaacttcga tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca
      721 tggaacgggt tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg
      781 cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta
      841 acggattcac cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc
      901 aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc
      961 gcatctcggg ctccttgcat gcaccattcc cgcttcctcg ctcactgact cgctacgctc
     1021 ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga
     1081 tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg
     1141 ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg
     1201 acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt
     1261 cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg
     1321 cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc
     1381 cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag
     1441 acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga
     1501 agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa
     1561 gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca
     1621 aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag
     1681 atcatcttat taatcagata aaatatttct agatttcagt gcaatttatc tcttcaaatg
     1741 tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg
     1801 cgcccaggat ctcaccgtca tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga
     1861 aggcgaagcg gcatgcataa tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc
     1921 tatgctactc cgtcaagccg tcaattgtct gattcgttac caattatgac aacttgacgg
     1981 ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg gccccggtgc
     2041 attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg cgaccgacgg
     2101 tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct
     2161 cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac agacgcgacg
     2221 gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct gccaggtgat
     2281 cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg agcgactcgt
     2341 taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc agcagctccg
     2401 aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg
     2461 gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac ggccagttaa
     2521 gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac cattcgcgag
     2581 cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa atatcacccg
     2641 gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga atggtgagat
     2701 tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg
     2761 cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc ggcagcaggg
     2821 gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa gaaaccaatt
     2881 gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc tcgctaacca
     2941 aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa gccatgacaa
     3001 aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat tatttgcacg
     3061 gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggat cctacctgac
     3121 gctttttatc gcaactctct actgtttctc catacccgtt ttttgggcta acaggaggaa
     3181 ttaaccatgg ttagcaaagg tgaagaactg tttaccggcg ttgtgccgat tctggtggaa
     3241 ctggatggtg atgtgaatgg ccataaattt agcgttcgtg gcgaaggcga aggtgatgcg
     3301 accaacggta aactgaccct gaaatttatt tgcaccaccg gtaaactgcc ggttccgtgg
     3361 ccgaccctgg tgaccaccct gacctatggc gttcagtgct ttagccgcta tccggatcat
     3421 atgaaacgcc atgatttctt taaaagcgcg atgccggaag gctatgtgca ggaacgtacc
     3481 attagcttca aagatgatgg cacctataaa acccgtgcgg aagttaaatt tgaaggcgat
     3541 accctggtga accgcattga actgaaaggt attgatttta aagaagatgg caacattctg
     3601 ggtcataaac tggaatataa tttcaacagc cataatgtgt atattaccgc cgataaacag
     3661 aaaaatggca tcaaagcgaa ctttaaaatc cgtcacaacg tggaagatgg tagcgtgcag
     3721 ctggcggatc attatcagca gaataccccg attggtgatg gcccggtgct gctgccggat
     3781 aatcattatc tgagcaccca gagcgttctg agcaaagatc cgaatgaaaa acgtgatcat
     3841 atggtgctgc tggaatttgt taccgccgcg ggcattaccc acggtatgga tgaactgtat
     3901 aaaggcagcc accatcatca tcaccattaa agctcgagat ctgcagctgg taccatatgg
     3961 gaattcgaag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa
     4021 atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt
     4081 cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg
     4141 gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga
     4201 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
     4261 atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac
     4321 gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt
     4381 ttgcgtttct acaaactctg tcgacacaga tgtaggtgtt ccacagggta gccagcagca
     4441 tcctgcgatg cagatccgga acataatggt gcagggcgct tgtttcggcg tgggtatggt
     4501 ggcaggcccc gtggccgggg gactgttggg cgctgccggc acctgtccta cgagttgcat
     4561 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     4621 gctaccggca gcggtgcgga ctgttgtaac tcagaataag aaatgaggcc gctcatggcg
     4681 ttctgttgcc cgtctcactg gtgaaaagaa aaacaaccct ggcgccgctt ctttgagcga
     4741 acgatcaaaa ataagtggcg ccccatcaaa aaaatattct caacataaaa aactttgtgt
     4801 aatacttgta acgctagatc tgggggacgg tccggcgacc agcgggtctc taaaacctag
     4861 catagcgggg ttcgacaccc cggtctctcg ccaactagta tccttagcga aagctaagga
     4921 ttttttttaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     4981 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagacgctc tcccttatgc
     5041 gactcctgca atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt
     5101 ttgacagctt atcatcgata agctttaatg cggtagttta tcacagttaa attgctaacg
     5161 cagtcaggca ccgtgtatga aatctaacaa tgcgctcatc gtcatcctcg gcaccgtcac
     5221 cctggatgct gtaggcatag gcttggttat gccggtactg ccgggcctct tgcgggatat
     5281 cgtccattcc gacagcatcg ccagtcacta tggcgtgctg ctagcgctat atgcgttgat
     5341 gcaatttcta tgcgcacccg ttctcggagc actgtccgac cgctttggcc gccgcccagt
     5401 cctgctcgct tcgctacttg gagccactat cgactacgcg atcatggcga ccacacccgt
     5461 cctgtggatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     5521 tgctggcgcc tatatcgccg acatcaccga t
//
