LOCUS       sequence-499716-        7714 bp    DNA     circular SYN 10-FEB-2026
DEFINITION  Site-specific incorporation of L-lactyl-lysine into target proteins 
            in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7714)
  AUTHORS   Shao C, Tang S, Yu S, Liu C, Zhang Y, Wan T, He Z, Yuan Q, Wu S, 
            Zhang H, Wan N, Zhan M, Tan RX, Hao H, Ye H, Wang N
  TITLE     Genetic code expansion reveals site-specific lactylation in living 
            cells reshapes protein functions.
  JOURNAL   Nat Commun. 2025 Jan 8;16(1):227. doi: 10.1038/s41467-024-55165-2.
  PUBMED    39779673
REFERENCE   2  (bases 1 to 7714)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 10, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-01-8"; volume: "16(1):227. doi"; pages: " 
            10.1038/s41467-024-55165-2"
COMMENT     Sequence Label: pNEU-KlacRS
FEATURES             Location/Qualifiers
     source          1..7714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(113..361)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(452..700)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(791..1039)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1087..1106)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1278..1657
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1658..1861
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1812..1832
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1906..1925
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1906..1924
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1944..1953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3341..3358)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3347..3571
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3617..4045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3704..3723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3914..3935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4054..4074)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4059..4388
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4239..4374
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4301..4320
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4455..5249
                     /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(4509..4528)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5119..5138
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5423..5544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5460..5479)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5514..5533
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5591..5607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5591..5607)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5604..5626)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5615..5631
                     /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(5639..5645,5646..5663,5664..5669))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5684..5705
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5822..5839)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5993..6581)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6073..6092)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6752..7543,7544..7612))
                     /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     7375..7394
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7613..7714,1..3))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcgcacattt ccccgaaaag tgccacctga cgtcgacaaa aaacggaaac cccgggaatc
       61 gaacccggct gaacggattt agagtccgtt cggtctccct gaccaggttt ccggtgtttc
      121 gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca
      181 tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact
      241 ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta
      301 tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct
      361 cttcctgccc ctcgacaaaa aacggaaacc ccgggaatcg aacccggctg aacggattta
      421 gagtccgttc ggtctccctg accaggtttc cggtgtttcg tcctttccac aagatatata
      481 aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac
      541 ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta
      601 ctaatatctt tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc
      661 gtatatgcaa atatgaagga atcatgggaa ataggccctc ttcctgcccc tcgacaaaaa
      721 acggaaaccc cgggaatcga acccggctga acggatttag agtccgttcg gtctccctga
      781 ccaggtttcc ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact
      841 ttcaagttac ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact
      901 acccaagaaa ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag
      961 tcaaattaat tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa
     1021 tcatgggaaa taggccctct tcctgcccct cgagtctaga cccgatcccc tatggtgcac
     1081 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     1141 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     1201 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     1261 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     1321 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     1381 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     1441 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1501 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     1561 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     1621 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     1681 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     1741 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     1801 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     1861 tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta
     1921 tagggagacc caagctggct agcgccacca tggataaaaa gcctttgaac actctgattt
     1981 ctgcgaccgg tctgtggatg tcccgcaccg gcaccatcca caaaatcaaa caccatgaag
     2041 ttagccgttc caaaatctac attgaaatgg cttgcggcga tcacctggtt gtcaacaact
     2101 cccgttcttc tcgtaccgct cgcgcactgc gccaccacaa atatcgcaaa acctgcaaac
     2161 gttgccgtgt tagcgatgag gacctgaaca aattcctgac caaagctaac gaggatcaga
     2221 cctccgtaaa agtgaaggta gtaagcgctc cgacccgtac taaaaaggct atgccaaaaa
     2281 gcgtggcccg tgccccgaaa cctctggaaa acaccgaggc ggctcaggct caaccatccg
     2341 gttctaaatt ttctccggcg atcccagtgt ccacccaaga atctgtttcc gtaccagcaa
     2401 gcgtgtctac cagcattagc agcatttcta ccggtgctac cgcttctgcg ctggtaaaag
     2461 gtaacactaa cccgattact agcatgtctg caccggtaca ggcaagcgcc ccagctctga
     2521 ctaaatccca gacggaccgt ctggaggtgc tgctgaaccc aaaggatgaa atctctctga
     2581 acagcggcaa gcctttccgt gagctggaaa gcgagctgct gtctcgtcgt aaaaaggatc
     2641 tgcaacagat ctacgctgag gaacgcgaga actatctggg taagctggag cgcgaaatta
     2701 ctcgcttctt cgtggatcgc ggtttcctgg agatcaaatc tccgattctg attccgctgg
     2761 aatacattga acgtatgggc atcgataatg ataccgaact gtctaaacag atcttccgtg
     2821 tggataaaaa cttctgtctg cgtccgatga tggccccgaa cctgctgaac tatgcgcgta
     2881 aactggaccg tgccctgccg gacccgatca aaattttcga gatcggtcct tgctaccgta
     2941 aagagtccga cggtaaagag cacctggaag aattcaccat gctgaacttc tttcagatgg
     3001 gtagcggttg cacgcgtgaa aacctggaat ccattatcac cgacttcctg aatcacctgg
     3061 gtatcgattt caaaattgtt ggtgacagct gtatggtgta tggcgatacg ctggatgtta
     3121 tgcacggcga tctggagctg tcttccgcag tagtgggccc aatcccgctg gatcgtgagt
     3181 ggggtatcga caaaccttgg atcggtgcgg gttttggtct ggagcgtctg ctgaaagtaa
     3241 aacacgactt caagaacatc aaacgtgctg cacgttccga gtcctattac aatggtattt
     3301 ctactaacct gtaagcggcc gctttaaacc cgctgatcag cctcgactgt gccttctagt
     3361 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3421 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3481 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3541 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     3601 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3661 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3721 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3781 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3841 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3901 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3961 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4021 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     4081 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     4141 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4201 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     4261 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     4321 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     4381 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     4441 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     4501 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     4561 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     4621 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     4681 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     4741 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     4801 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     4861 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     4921 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4981 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     5041 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     5101 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     5161 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     5221 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     5281 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     5341 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     5401 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     5461 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5521 gtccaaactc atcaatgtat cttatcatgt ctgtataccg gtcctctagc tagagcttgg
     5581 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5641 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5701 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5761 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5821 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5881 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5941 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6001 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6061 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6121 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6181 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6241 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6301 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6361 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6421 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6481 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6541 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6601 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6661 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6721 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6781 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6841 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6901 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6961 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7021 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7081 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7141 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7201 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7261 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7321 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7381 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7441 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7501 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7561 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7621 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7681 aatgtattta gaaaaataaa caaatagggg ttcc
//
