LOCUS       sequence-332393-        7885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes for MmPyl tRNA synthetase/tRNA pair and for GFP reporter 
            with TAG mutation at site 35, for unnatural amino acid incorporation
            into the GFP protein in Vibrio natriegens..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7885)
  AUTHORS   Ozer E, Alfonta L
  TITLE     Genetic Code Expansion of Vibrio natriegens.
  JOURNAL   Front Bioeng Biotechnol. 2021 Feb 26;9:594429. doi: 
            10.3389/fbioe.2021.594429. eCollection 2021.
  PUBMED    33718334
REFERENCE   2  (bases 1 to 7885)
  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: "Front 
            Bioeng Biotechnol."; date: "2021-02-26"; volume: "9:594429. doi"; 
            pages: " 10.3389/fbioe.2021.594429. eCollection 2021"
COMMENT     Sequence Label: pRaGE Pyl TAG GFP Y35TAG
FEATURES             Location/Qualifiers
     source          1..7885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     111..129
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1693..1909
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1910..2710
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPAAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTEEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     RBS             5114..5136
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5155..5172
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5209..5230)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5470..5489)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5817..5838
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6033..6050)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6204..6792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6284..6303)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6963..7754,7755..7823))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7586..7605
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7824..7885,1..43))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
       61 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
      121 tcacgaggcc ctttcgtctt caagaattct ggcgaatcct ctgaccagcc agaaaacgac
      181 ctttctgtgg tgaaaccgga tgctgcaatt cggatccgcg aataccgctt ccacaaacat
      241 tgctcaaaag tatctctttg ctatatatct ctgtgctata tccctatata acctacccat
      301 ccacctttcg ctccttgaac ttgcatctaa actcgacctc tacatttttt atgtttatct
      361 ctagtattac tctttagaca aaaaaattgt agtaagaact attcatagag tgaatcgaaa
      421 acaatacgaa aatgtaaaca tttcctatac gtagtatata gagacaaaat agaagaaacc
      481 gttcataatt ttctgaccaa tgaagaatca tcaacgctat cactttctgt tcacaaagta
      541 tgcgcaatcc acatcggtat agaatataat cggggatgcc tttatcttga aaaaatgcac
      601 ccgcagcttc gctagtaatc agtaaacgcg ggaagtggag tcaggctttt tttatggaag
      661 agaaaataga caccaaagta gccttcttct aaccttaacg gacctacagt gcaaaaagtt
      721 atcaagagac tgcattatag agcgcacaaa ggagaaaaaa agtaatctaa gatgctttgt
      781 tagaaaaata gcgctctcgg gatgcatttt tgtagaacaa aaaagaagta tagattcttt
      841 gttggtaaaa tagcgctctc gcgttgcatt tctgttctgt aaaaatgcag ctcagattct
      901 ttgtttgaaa aattagcgct ctcgcgttgc atttttgttt tacaaaaatg aagcacagat
      961 tcttcgttgg taagatagcg ctttcgcgtt gcatttctgt tctgtaaaaa tgcagctcag
     1021 attctttgtt tgaaaagtta gcgctctcgc gttgcatttt tgttctacaa aatgaagcac
     1081 agatgcttcg ttaacaaaga tatgctattg aagtgcaaga tggaaacgca gaaaatgaac
     1141 cggggatgcg acgtgcaaga ttacctatgc aatagatgca atagtttctc caggaaccga
     1201 aatacataca ttgtcttccg taaagcgcta gactatatat tattatacag gttcaaatat
     1261 actatctgtt tcagggaaaa ctcccaggtt cggatgttca aaattcaatg atgggtaaca
     1321 agtacgatcg taaatctgta aaacagtttg tcggatatta ggctgtatct cctcaaagcg
     1381 tattcgaata tcattgagaa gctgcagcgt cacatcggat aataatgttg gcagccattg
     1441 tagaagtgcc ttttgcattt ctagtctctt tctcggtcta gctagtttta ctacatcgcg
     1501 aagatagaat cttagatcac actgcctttg ctgagctgga tcaatagagt aacaaaagag
     1561 tggtaaggct tcgttaaagg acaaggacct gagcggaagt gtatcgtaca gtagacggag
     1621 tatactaggt atagtctata gtccgtggaa ttaattctca tgtttgacag cttatcatcg
     1681 ataagctagc ttttcaattc atcatttttt tttttattct tttttttgat ttcggtttcc
     1741 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
     1801 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
     1861 cttaacccag ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
     1921 tacatataag gaacgtgctg ctactcatcc tagtcctgct gctgccaagc tatttaatat
     1981 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
     2041 actggagtta gttgaggcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
     2101 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
     2161 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
     2221 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
     2281 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa cagaggaacc
     2341 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagagta
     2401 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
     2461 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ctgattatga cacccggtgt
     2521 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     2581 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     2641 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     2701 ccagcaaaac ggatccgctt aaaccttact gcaagggtaa ggtcaaggtt tgttatcaat
     2761 gaagttaatt tcgttgaacc ccgcatcgag aggttatttg ggtatatgaa cacggaagga
     2821 ttgtagtatt gacttacgat ggtaaaatgt cgccactttt atccttgaca atacaacgag
     2881 aaccctctcg ttgaacatta accctaagga actcaaatgg ataaaaaacc actaaacact
     2941 ctgatctctg ctactggtct gtggatgagt cgtaccggaa ccattcataa aatcaaacac
     3001 cacgaggtta gccgttcgaa aatctatatt gagatggcgt gtggcgatca tctggttgtg
     3061 aacaatagcc gctcttctcg tacagcacgt gcactgcgtc accacaaata tcgtaaaacc
     3121 tgtaaacgtt gccgtgtgtc cgatgaggat ctgaacaaat tcctgacaaa agccaatgag
     3181 gaccaaacaa gcgtgaaagt gaaagtcgtt agcgctccta cccgtactaa aaaagcaatg
     3241 ccgaaatccg ttgctcgtgc ccctaaacca ctggaaaaca ctgaagcagc acaggcacag
     3301 ccgtctggaa gcaaattctc tccggccatt cctgtttcta cccaggagtc cgtttctgtt
     3361 ccagcaagtg tgagcaccag cattagcagt attagcaccg gtgccaccgc tagcgccctg
     3421 gttaaaggca ataccaatcc gattacaagc atgtctgccc cggttcaagc atcagctcca
     3481 gcactgacaa aatcccaaac cgatcgtctg gaggttctgc tgaatccgaa agacgaaatc
     3541 agcctgaatt ccggcaaacc gtttcgtgaa ctggagagcg aactgctgtc acgtcgtaaa
     3601 aaagacctgc aacaaatcta tgccgaagaa cgtgagaact atctggggaa actggaacgt
     3661 gaaatcaccc gctttttcgt ggatcgtggc tttctggaga tcaaatcccc gattctgatt
     3721 cctctggagt atatcgagcg tatgggcatc gacaatgata ccgaactgag caaacaaatt
     3781 ttccgtgtgg ataaaaactt ctgtctgcgc cctatgctgg caccaaatct gtataactat
     3841 ctgcgcaaac tggaccgtgc cctgcctgat cctatcaaaa tcttcgagat cggcccgtgt
     3901 tatcgtaaag agtccgacgg taaagaacat ctggaggagt ttaccatgct gaacttttgc
     3961 caaatgggtt caggttgtac tcgtgagaac ctggaaagca tcatcaccga ttttctgaac
     4021 cacctgggca ttgacttcaa aattgtgggc gacagctgta tggtgtatgg cgacaccctg
     4081 gatgtcatgc acggcgacct ggaactgtct agtgccgttg ttggaccaat tccgctggac
     4141 cgtgagtggg gtatcgacaa accgtggatc ggagcaggat tcggtctgga acgcctgctg
     4201 aaagtgaaac acgacttcaa aaacatcaaa cgtgccgccc gttctgaatc gtattataac
     4261 gggatttcta ccaacctgta attcaattgc atagatattc aaaaaccgcg gtaatttccg
     4321 cggttttttt atgtctaaag aatgtaataa aagcgaaagt agtgaacaga tttgactgat
     4381 gtacatgcta tgtcggcgtg ttgatgtttg ttttttgata aatttttgta attaaagatc
     4441 gtggttttta gtgcttattc aatggctgat tttcaaacta tccctctgat taatatctac
     4501 tatctataaa gttccccaag tggcatctca atagtgagac aaatatctgc attagttact
     4561 ttgttgtgac ttttggcgca atactcacga tgagtgtcca cacagattga tatggtttat
     4621 gaagtttaag agatagaaca tcccccaaga tgttcaactt agtggaaacc tgatcatgta
     4681 gatcgaatgg actctaaatc cgttcagccg ggttagattc ccggggtttc cgccattctc
     4741 taagcatttt tggaataaaa cgtccaaacc agagcaagca attgacactg gttggcattt
     4801 ttgactctga aagtctttag aaaatgtact tcctcgtgaa gaaacatagc tctttaacag
     4861 agcggcagca agtgggggac agcagaagac ctgaccgccg cagagtggat gtttgacatg
     4921 gtgaagacta tcgcaccatc agccagaaaa ccgaattttg ctgggtgggc taacgatatc
     4981 cgcctgatgc gtgaacgtga cggacgtaac caccgcgaca tgtgtgtgct gttccgctgg
     5041 gcatgctgag ctaacaccgt gcgtgtagac aattttacct ctggcggtga taatggttgc
     5101 agctagcaat aattttgttt aactttaaga aggagatata ccatgggcag cagccatcat
     5161 catcatcatc acagctctag agagcttttc actggcgttg ttcccatcct ggtcgagctg
     5221 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     5281 tagggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     5341 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     5401 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     5461 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     5521 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     5581 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     5641 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     5701 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     5761 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     5821 gtcctgctgg agttcgtgac cgccgccggg atctgactcg agcaaagccc gccgaaaggc
     5881 gggcttttct gtgtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     5941 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     6001 taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     6061 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6121 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6181 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6241 aatcgatgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6301 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6361 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6421 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6481 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6541 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6601 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6661 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6721 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6781 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6841 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6901 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6961 agtcaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     7021 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     7081 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7141 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7201 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7261 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7321 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7381 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7441 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7501 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7561 tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg
     7621 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7681 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7741 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7801 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7861 ggttattgtc tcatgagcgg ataca
//
