LOCUS       sequence-100911-        7315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains GFP tag for direct live-cell fluorescence imaging of the 
            resofurin ligase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7315)
  AUTHORS   Liu DS, Nivon LG, Richter F, Goldman PJ, Deerinck TJ, Yao JZ, 
            Richardson D, Phipps WS, Ye AZ, Ellisman MH, Drennan CL, Baker D, 
            Ting AY
  TITLE     Computational design of a red fluorophore ligase for site-specific 
            protein labeling in living cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Oct 28;111(43):E4551-9. doi: 
            10.1073/pnas.1404736111. Epub 2014 Oct 13.
  PUBMED    25313043
REFERENCE   2  (bases 1 to 7315)
  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: 
            "10.1073/pnas.1404736111"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2014-10-28- 28"; volume: "111"; issue: "43"; pages: "E4551-9"
COMMENT     Sequence Label: pcDNA3-GFP-LplA(AAG)
FEATURES             Location/Qualifiers
     source          1..7315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(952..954,955..957,958..1668)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(952..954,955..957,958..1668)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(997..1018)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1258..1277)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1605..1626
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1678..1701
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2867..2885)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2868..2885)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2905..2922)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2911..3135
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3181..3609
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3268..3287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3478..3499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3618..3638)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3623..3953
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3804..3939
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3866..3885
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4020..4814
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4074..4093)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4684..4703
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4988..5109
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5025..5044)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5079..5098
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5158..5174)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5158..5174)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5171..5193)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5182..5198
                     /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)."
     promoter        complement(join(5206..5212,5213..5230,5231..5236))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5251..5272
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5389..5406)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5560..6148)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5640..5659)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6319..7110,7111..7179))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6942..6961
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7180..7284)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catggtgagc
      961 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1021 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1081 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1141 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1201 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1261 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1321 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1381 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1441 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1501 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1561 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1621 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagaa ggatccagac
     1681 tacaaggatg acgacgataa gatgagcacc ctgagactgc tgatcagcga cagctacgac
     1741 ccctggttca acctggccgt ggccgagtgc atcttcagac agatgcccgc cacccagaga
     1801 gtgctgttcc tgtggagaaa cgccgacacc gtggtgatcg gcagagccca gaacccctgg
     1861 aaggagtgca acaccagaag aatggaggag gacaacgtga gactggccag aagaagcagc
     1921 ggcggcggcg ccgtgttcca cgacctgggc aacacctgct tcaccttcat ggccggcaag
     1981 cccgagtacg acaagaccat cagcaccagc atcgtgctga acgccctgaa cgccctgggc
     2041 gtgagcgccg aggccagcgg cagaaacgac ctggtggtga agaccgtgga gggcgacaga
     2101 aaggtgagcg gcagcgccta cagagagacc aaggacagag gcgcccacgg cggcaccctg
     2161 ctgctgaacg ccgacctgag cagactggcc aactacctga accccgacaa gaagaagctg
     2221 gccgccaagg gcatcaccag cgtgagaagc agagtgacca acctgaccga gctgctgccc
     2281 ggcatcaccc acgagcaggt gtgcgaggcc atcaccgagg ccttcttcgc ccactacggc
     2341 gagagagtgg aggccgagat catcagcccc aacaagaccc ccgacctgcc caacttcgcc
     2401 gagaccttcg ccagacagag cagctgggag tggaacttcg gccaggcccc cgccttcagc
     2461 cacctgctgg acgagagatt cacctggggc ggcgtggagc tgcacttcga cgtggagaag
     2521 ggccacatca ccagagccca ggtgttcacc gacagcctga accccgcccc cctggaggcc
     2581 ctggccggca gactgcaggg ctgcctgtac agagccgaca tgctgcagca ggagtgcgag
     2641 gccctgctgg tggacttccc cgagcaggag aaggagctga gagagctgag cgcctggatg
     2701 gccggcgccg tgagataaga attctgcaga tatccatcac actggcggcc gctcgagcgc
     2761 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccaacgcca tctgatcgtt
     2821 ggcaaccagc atcgcagtgg gaacgatgcc ctctagaggg ccctattcta tagtgtcacc
     2881 taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2941 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3001 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3061 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3121 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     3181 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3241 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3301 cgttcgccgg ctttccccgt caagctctaa atcggggcat ccctttaggg ttccgattta
     3361 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3421 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3481 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3541 aagggatttt ggggatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3601 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3661 aggcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3721 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3781 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3841 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3901 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3961 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     4021 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4081 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4141 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4201 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4261 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4321 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4381 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4441 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4501 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4561 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4621 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4681 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4741 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4801 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4861 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4921 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4981 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5041 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5101 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     5161 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5221 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5281 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5341 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5401 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5461 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5521 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5581 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5641 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5701 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
     5761 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5821 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5881 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5941 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6001 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6061 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6121 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6181 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6241 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6301 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6361 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6421 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6481 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6541 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6601 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6661 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6721 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6781 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6841 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6901 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6961 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7021 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7081 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7141 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7201 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7261 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
