LOCUS       sequence-261547-        5978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses sfGFP with an unnatural amino acid at the 150th position 
            in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5978)
  AUTHORS   Williams TL, Iskandar DJ, Nodling AR, Tan Y, Luk LYP, Tsai YH
  TITLE     Transferability of N-terminal mutations of pyrrolysyl-tRNA 
            synthetase in one species to that in another species on unnatural 
            amino acid incorporation efficiency.
  JOURNAL   Amino Acids. 2021 Jan;53(1):89-96. doi: 10.1007/s00726-020-02927-z. 
            Epub 2020 Dec 17.
  PUBMED    33331978
REFERENCE   2  (bases 1 to 5978)
  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: 
            "10.1007/s00726-020-02927-z"; journalName: "Amino Acids"; date: 
            "2021-01"; volume: "53"; issue: "1"; pages: "89-96"
COMMENT     Sequence Label: pET sfGFP(150TAG)
FEATURES             Location/Qualifiers
     source          1..5978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             811..828
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             840..857
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(910..928)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      924..971
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1008..1463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1095..1114)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1305..1326
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1555..2370)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1555..2370)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2278..2297
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2362..2381
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2492..3080
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2981..3000
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3234..3251
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3266..3405
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3331..3350)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3491..3513
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3507..3698)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4507..5592)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSGA*KVLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASP
                     RALADSLMQLARQVSRLESGQ"
     primer_bind     5554..5573
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5593..5670)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5876..5895
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gttagcaaag gtgaagaact gtttaccggc
      121 gttgtgccga ttctggtgga actggatggt gatgtgaatg gccataaatt tagcgttcgt
      181 ggcgaaggcg aaggtgatgc gaccaacggt aaactgaccc tgaaatttat ttgcaccacc
      241 ggtaaactgc cggttccgtg gccgaccctg gtgaccaccc tgacctatgg cgttcagtgc
      301 tttagccgct atccggatca tatgaaacgc catgatttct ttaaaagcgc gatgccggaa
      361 ggctatgtgc aggaacgtac cattagcttc aaagatgatg gcacctataa aacccgtgcg
      421 gaagttaaat ttgaaggcga taccctggtg aaccgcattg aactgaaagg tattgatttt
      481 aaagaagatg gcaacattct gggtcataaa ctggaatata atttcaacag ccattaggtg
      541 tatattaccg ccgataaaca gaaaaatggc atcaaagcga actttaaaat ccgtcacaac
      601 gtggaagatg gtagcgtgca gctggcggat cattatcagc agaatacccc gattggtgat
      661 ggcccggtgc tgctgccgga taatcattat ctgagcaccc agagcgttct gagcaaagat
      721 ccgaatgaaa aacgtgatca tatggtgctg ctggaatttg ttaccgccgc gggcattacc
      781 cacggtatgg atgaactgta taaaggcagc caccatcatc atcaccatta aagctcgagc
      841 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
      901 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
      961 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     1021 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1081 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1141 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1201 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1261 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1321 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1381 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1441 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     1501 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     1561 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     1621 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     1681 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     1741 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     1801 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     1861 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     1921 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     1981 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     2041 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     2101 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     2161 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     2221 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     2281 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     2341 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     2401 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2461 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2521 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2581 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2641 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2701 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2761 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2821 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2881 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2941 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3001 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3061 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3121 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3181 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3241 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3301 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3361 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3421 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3481 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3541 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3601 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3661 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3721 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3781 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     3841 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     3901 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3961 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     4021 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     4081 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     4141 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     4201 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4261 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     4321 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4381 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4441 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4501 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4561 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4621 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcac
     4681 cttttaggca ccgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     4741 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     4801 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     4861 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     4921 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     4981 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5041 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     5101 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     5161 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5221 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5281 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5341 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5401 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5461 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     5521 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     5581 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     5641 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     5701 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     5761 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     5821 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     5881 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     5941 gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
