LOCUS       sequence-495655-        7652 bp    DNA     circular SYN 14-JAN-2026
DEFINITION  Constructs used for LplA-catalyzed labeling in mammalian cells. LplA
            is expressed as a fusion with mCherry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7652)
  AUTHORS   Cavanagh PE, Xue AG, Dai SA, Qiang A, Matsui T, Ting AY
  TITLE     Computational design of conformation-biasing mutations to alter 
            protein functions.
  JOURNAL   Science. 2026 Jan 8:eadv7953. doi: 10.1126/science.adv7953.
  PUBMED    41505504
REFERENCE   2  (bases 1 to 7652)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 14, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2026 Jan 8:eadv7953. doi: 10.1126/science.adv7953."
COMMENT     Sequence Label: His6-mCherry-Flag-LplAW37V and C55I
FEATURES             Location/Qualifiers
     source          1..7652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    7..128
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    137..725
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             243..254
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     652..672
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1765..1788)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1798..2505)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2073..2092)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2342..2360
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2518..2535)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2547..2556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2575..2594)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2576..2594)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2639..2842)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2668..2688)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2843..3222
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3394..3413
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3488..3592
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3593..3661,3662..4453)
                     /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     complement(3811..3830)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4624..5212
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5113..5132
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5366..5383
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5500..5521
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5536..5541,5542..5559,5560..5566)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5574..5590
                     /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)."
     primer_bind     5579..5601
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5598..5614
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5598..5614
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5663..5784
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5958..6752)
                     /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(6069..6088)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6679..6698
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6819..7148)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6833..6968
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6887..6906)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     7133..7153
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(7162..7590)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7272..7293)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7484..7503
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 caattgtaag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
       61 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
      121 aacaagtttt aattaagcgg ggaggcggcc caaagggaga tccgactcgt ctgagggcga
      181 aggcgaagac gcggaagagg ccgcagagcc ggcagcaggc cgcgggaagg aaggtccgct
      241 ggattgaggg ccgaagggac gtagcagaag gacgtcccgc gcagaatcca ggtggcaaca
      301 taggcgagca gccaaggaaa ggacgatgat ttccccgaca acaccacgga attgtcagtg
      361 cccaacagcc gagcccctgt ccagcagcgg gcaaggcagg cggcgatgag ttccgccgtg
      421 gcaataggga gggggaaagc gaaagtcccg gaaaggagct gacaggtggt ggcaatgccc
      481 caaccagtgg gggttgcgtc agcaaacaca gtgcacacca cgccacgttg cctgacaacg
      541 ggccacaact cctcataaag agacagcaac caggatttat acaaggagga gaaaatgaaa
      601 gccatacggg aagcaatagc atgatacaaa ggcattaaag cagcgtatcc acatagcgta
      661 aaaggagcaa catagttaag aataccagtc aatctttcac aaattttgta atccagaggt
      721 tgatttcgcg agatatctgc agaattctta tctcacggcg ccggccatcc aggcgctcag
      781 ctctctcagc tccttctcct gctcggggaa gtccaccagc agggcctcgc actcctgctg
      841 cagcatgtcg gctctgtaca ggcagccctg cagtctgccg gccagggcct ccaggggggc
      901 ggggttcagg ctgtcggtga acacctgggc tctggtgatg tggcccttct ccacgtcgaa
      961 gtgcagctcc acgccgcccc aggtgaatct ctcgtccagc aggtggctga aggcgggggc
     1021 ctggccgaag ttccactccc agctgctctg tctggcgaag gtctcggcga agttgggcag
     1081 gtcgggggtc ttgttggggc tgatgatctc ggcctccact ctctcgccgt agtgggcgaa
     1141 gaaggcctcg gtgatggcct cgcacacctg ctcgtgggtg atgccgggca gcagctcggt
     1201 caggttggtc actctgcttc tcacgctggt gatgcccttg gcggccagct tcttcttgtc
     1261 ggggttcagg tagttggcca gtctgctcag gtcggcgttc agcagcaggg tgccgtggtg
     1321 gaagcctctg tccttggtct ctctgtaggc gctgccgctc acctttctgt cgccctccac
     1381 ggtcttcacc accaggtcgt ttctgccgct ggcctcggcg ctcacgccca gggcgttcag
     1441 ggcgttcagc acgatgctgg tgctgatggt cttgtcgtac tcgggcttgc cggccatgaa
     1501 ggtgaagcag gtgttgccca ggtcgtggaa cacggcgccg ccgccgctgc ttcttctggc
     1561 cagtctcacg ttgtcctcct ccattcttct ggtgttgatc tccttccagg ggttctgggc
     1621 tctgccgatc accacggtgt cggcgtttct caccaggaac agcactctct gggtggcggg
     1681 catctgtctg aagatgcact cctccacggc caggttgaac caggggtcgt agctgtcgct
     1741 gatcagcagt ctcagggtgc tcatcttatc gtcgtcatcc ttgtagtctg gatccttctt
     1801 gtacagctcg tccatgccgc cggtggagtg gcggccctcg gcgcgttcgt actgttccac
     1861 gatggtgtag tcctcgttgt gggaggtgat gtccaacttg atgttgacgt tgtaggcgcc
     1921 gggcagctgc acgggcttct tggccttgta ggtggtcttg acctcagcgt cgtagtggcc
     1981 gccgtccttc agcttcagcc tctgcttgat ctcgcccttc agggcgccgt cctcggggta
     2041 catccgctcg gaggaggcct cccagcccat ggtcttcttc tgcattacgg ggccgtcgga
     2101 ggggaagttg gtgccgcgca gcttcacctt gtagatgaac tcgccgtcct gcagggagga
     2161 gtcctgggtc acggtcacca cgccgccgtc ctcgaagttc atcacgcgct cccacttgaa
     2221 gccctcgggg aaggacagct tcaagtagtc ggggatgtcg gcggggtgct tcacgtaggc
     2281 cttggagccg tacatgaact gaggggacag gatgtcccag gcgaagggca gggggccacc
     2341 cttggtcacc ttcagcttgg cggtctgggt gccctcgtag gggcggccct cgccctcgcc
     2401 ctcgatctcg aactcgtggc cgttcacgga gccctccatg tgcaccttga agcgcatgaa
     2461 ctccttgatg atggccatgt tatcctcctc gcccttgctc accatgctag ccataccatg
     2521 atgatgatga tgatgagaac cccgcaccat ggtggcggcc gcaagcttgg gtctccctat
     2581 agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt agccagagag
     2641 ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg
     2701 agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga
     2761 cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga
     2821 tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc
     2881 aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt
     2941 cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg
     3001 cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta
     3061 tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg
     3121 ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc
     3181 ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta tatctggccc
     3241 gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg caattgtcgg
     3301 tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg acctccaaca
     3361 cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt gtactgagag
     3421 tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca cttttcgggg
     3481 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3541 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3601 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3661 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3721 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3781 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3841 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3901 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3961 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4021 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     4081 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     4141 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4201 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4261 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     4321 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4381 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4441 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4501 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4561 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4621 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4681 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4741 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4801 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4861 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4921 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4981 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5041 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5101 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5161 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5221 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5281 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5341 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     5401 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     5461 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     5521 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     5581 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc tctagctaga
     5641 ggtcgacggt atacagacat gataagatac attgatgagt ttggacaaac cacaactaga
     5701 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     5761 attataagct gcaataaaca agttggggtg ggcgaagaac tccagcatga gatccccgcg
     5821 ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa
     5881 ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc
     5941 gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
     6001 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
     6061 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
     6121 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
     6181 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg
     6241 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
     6301 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
     6361 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
     6421 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
     6481 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg
     6541 gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg
     6601 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
     6661 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga
     6721 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     6781 tcagatccga aaatggatat acaagctccc gggagctttt tgcaaaagcc taggcctcca
     6841 aaaaagcctc ctcactactt ctggaatagc tcagaggcag aggcggcctc ggcctctgca
     6901 taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg
     6961 ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt
     7021 tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg
     7081 agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac
     7141 tgacacacat tccacagaat taattcgcgt taaatttttg ttaaatcagc tcatttttta
     7201 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     7261 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     7321 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     7381 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     7441 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     7501 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     7561 ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc cctagagccc cagctggttc
     7621 tttccgcctc agaagccata gagcccaccg ca
//
