LOCUS       sequence-392921-        5952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  superfolder GFP with N150TAA mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5952)
  AUTHORS   Bednar RM, Jana S, Kuppa S, Franklin R, Beckman J, Antony E, Cooley 
            RB, Mehl RA
  TITLE     Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino
            Acids That Enable Efficient Protein Dual-Labeling in Cells.
  JOURNAL   ACS Chem Biol. 2021 Nov 19;16(11):2612-2622. doi: 
            10.1021/acschembio.1c00649. Epub 2021 Sep 30.
  PUBMED    34590824
REFERENCE   2  (bases 1 to 5952)
  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: 
            "10.1021/acschembio.1c00649"; journalName: "ACS Chem Biol"; date: 
            "2021-11-19- 19"; volume: "16"; issue: "11"; pages: "2612-2622"
COMMENT     Sequence Label: pET28-sfGFP[N150TAA]
FEATURES             Location/Qualifiers
     source          1..5952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      24..479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(111..130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     321..342
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(571..1386)
                     /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(571..1386)
                     /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     1294..1313
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1378..1397
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1508..2096
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1997..2016
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2250..2267
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2282..2421
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2347..2366)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2507..2529
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2523..2714)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3523..4605)
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4567..4586
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4606..4683)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4889..4908
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4992..5011
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4992..5010
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5011..5035
                     /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             5050..5072
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5808..5825
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5878..5896)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5892..5939
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
       61 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      121 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
      181 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
      241 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
      301 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
      361 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
      421 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
      481 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
      541 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
      601 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
      661 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
      721 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
      781 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
      841 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
      901 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
      961 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     1021 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     1081 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     1141 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     1201 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     1261 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     1321 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     1381 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     1441 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1501 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     1561 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1621 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     1681 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1741 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1801 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1861 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1921 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1981 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2041 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2101 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2161 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2221 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2281 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     2341 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     2401 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2461 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     2521 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     2581 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     2641 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     2701 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     2761 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     2821 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     2881 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     2941 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     3001 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     3061 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     3121 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     3181 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     3241 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     3301 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     3361 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     3421 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     3481 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     3541 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3601 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     3661 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     3721 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     3781 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     3841 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     3901 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     3961 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     4021 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     4081 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     4141 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     4201 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     4261 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     4321 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     4381 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     4441 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     4501 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     4561 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     4621 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     4681 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     4741 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     4801 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     4861 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     4921 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     4981 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     5041 agaaataatt ttgtttaact ttaagaagga gatataccat ggttagcaaa ggtgaagaac
     5101 tgtttaccgg cgttgtgccg attctggtgg aactggatgg tgatgtgaat ggccataaat
     5161 ttagcgttcg tggcgaaggc gaaggtgatg cgaccaacgg taaactgacc ctgaaattta
     5221 tttgcaccac cggtaaactg ccggttccgt ggccgaccct ggtgaccacc ctgacctatg
     5281 gcgttcagtg ctttagccgc tatccggatc atatgaaacg ccatgatttc tttaaaagcg
     5341 cgatgccgga aggctatgtg caggaacgta ccattagctt caaagatgat ggcacctata
     5401 aaacccgtgc ggaagttaaa tttgaaggcg ataccctggt gaaccgcatt gaactgaaag
     5461 gtattgattt taaagaagat ggcaacattc tgggtcataa actggaatat aatttcaaca
     5521 gccattaagt gtatattacc gccgataaac agaaaaatgg catcaaagcg aactttaaaa
     5581 tccgtcacaa cgtggaagat ggtagcgtgc agctggcgga tcattatcag cagaataccc
     5641 cgattggtga tggcccggtg ctgctgccgg ataatcatta tctgagcacc cagagcgttc
     5701 tgagcaaaga tccgaatgaa aaacgtgatc atatggtgct gctggaattt gttaccgccg
     5761 cgggcattac ccacggtatg gatgaactgt ataaaggcag cctcgagcac caccaccacc
     5821 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     5881 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     5941 tgaaaggagg aa
//
