LOCUS       sequence-154222-        7278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP fusion to levoglucosan kinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7278)
  AUTHORS   Wrenbeck EE, Bedewitz MA, Klesmith JR, Noshin S, Barry CS, Whitehead
            TA
  TITLE     An Automated Data-Driven Pipeline for Improving Heterologous Enzyme 
            Expression.
  JOURNAL   ACS Synth Biol. 2019 Mar 15;8(3):474-481. doi: 
            10.1021/acssynbio.8b00486. Epub 2019 Feb 8.
  PUBMED    30721031
REFERENCE   2  (bases 1 to 7278)
  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/acssynbio.8b00486"; journalName: "ACS Synth Biol"; date: 
            "2019-03-15- 15"; volume: "8"; issue: "3"; pages: "474-481"
COMMENT     Sequence Label: pET29NK-LGK/mGFPmut3
FEATURES             Location/Qualifiers
     source          1..7278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(560..1375)
                     /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(560..1375)
                     /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     1283..1302
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1367..1386
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1986..2005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2239..2256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2271..2413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2339..2358)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2499..2521
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /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     4559..4578
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4598..4675)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4881..4900
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4988..5007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4988..5006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5007..5031
                     /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             5046..5068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6402..7109
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
                     CTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
                     KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFV
                     TAAGITHGMDELYK"
     CDS             6402..7109
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
                     CTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
                     KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFV
                     TAAGITHGMDELYK"
     primer_bind     complement(6426..6454)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     7049..7069
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             7110..7127
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7192..7210)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7206..7253
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
     2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3781 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc
     4981 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     5041 ataattttgt ttaactttaa gaaggagata tacatatgcc gattgcgacc tcaacgggtg
     5101 ataatgttct ggactttacg gttctgggcc tgaatagcgg tacgagtatg gatggtattg
     5161 actgcgcact gtgtcatttc tatcagaaaa ccccggatgc tccgatggaa tttgaactgc
     5221 tggaatacgg cgaagttccg ctggcgcagc cgattaaaca acgtgtcatg cgcatgatcc
     5281 tggaagatac cacgagcccg tctgaactgt cagaagtcaa cgtgattctg ggtgaacatt
     5341 ttgcggatgc cgtccgtcag ttcgcggccg aacgcaatgt ggatctgtca accattgacg
     5401 caatcgcttc gcacggccag acgatttggc tgctgagtat gccggaagaa ggtcaagtga
     5461 aatccgccct gaccatggca gaaggcgcta tcctggcgag tcgtacgggt attacctcca
     5521 tcacggattt ccgtatttcc gaccaggcag ctggtcgtca aggtgcaccg ctgatcgcat
     5581 ttttcgatgc tctgctgctg catcacccga ccaaactgcg cgcgtgccag aacattggcg
     5641 gtatcgccaa tgtgtgtttt attccgccgg atgttgacgg ccgtcgcacc gatgaatatt
     5701 acgattttga cacgggtccg ggcaacgtgt tcatcgacgc agtggttcgt cattttacca
     5761 atggtgaaca ggaatatgat aaagacggtg ctatgggcaa acgcggtaaa gtcgatcagg
     5821 aactggtgga tgactttctg aaaatgccgt atttccaact ggacccgccg aaaaccacgg
     5881 gccgtgaagt ttttcgcgat accctggcac atgacctgat tcgtcgcgcg gaagccaaag
     5941 gtctgagccc ggatgacatc gtggccacca cgacccgtat tacggcacag gctatcgttg
     6001 atcactatcg tcgctacgcg ccgtcacaag aaattgacga aatcttcatg tgcggcggtg
     6061 gcgcctataa cccgaatatt gtggaattta tccagcaatc gtacccgaac accaaaatta
     6121 tgatgctgga tgaagcaggt gtcccggcag gtgcaaaaga agcgattacg ttcgcctggc
     6181 agggcatgga agccctggtt ggtcgtagca tcccggttcc gacccgtgtc gaaacgcgcc
     6241 agcactatgt gctgggcaaa gttagcccgg gtctgaatta ccgctctgtg atgaaaaaag
     6301 gcatggcatt tggtggcgat gctcagcaac tgccgtgggt ttctgaaatg atcgtgaaga
     6361 aaaaaggcaa agttatcacc aacaactggg cgctcgagcg taaaggagaa gaacttttca
     6421 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg
     6481 tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca
     6541 ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttcggt tatggtgttc
     6601 aatgctttgc gagataccca gatcacatga aacagcatga ctttttcaag agtgccatgc
     6661 ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac
     6721 gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta aaaggtattg
     6781 attttaaaga agatggaaac attcttggac acaaattgga atacaactat aactcacaca
     6841 atgtatacat catggcagac aaacaaaaga atggaatcaa agttaacttc aaaattagac
     6901 acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat actccaattg
     6961 gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct aaactttcga
     7021 aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga
     7081 ttacacatgg catggatgaa ctatacaaac accatcacca tcaccattaa cctcagcaat
     7141 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     7201 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     7261 gaggaactat atccggat
//
