LOCUS       sequence-476296-        7865 bp    DNA     circular SYN 18-JUN-2025
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 7865)
  TITLE     Genetic code expansion reveals site-specific lactylation in living 
            cells reshapes protein functions
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7865)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pNEU-KlacRS
FEATURES             Location/Qualifiers
     source          1..7865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(112..360)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(450..698)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(788..1036)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(1126..1374)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1422..1441)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1613..1992
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1993..2196
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2147..2167
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2241..2260
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2241..2259
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3492..3509)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3498..3722
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3768..4196
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3855..3874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4065..4086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4205..4225)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4210..4539
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4390..4525
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4452..4471
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4606..5400
                     /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(4660..4679)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5270..5289
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5574..5695
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5611..5630)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5665..5684
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5742..5758)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5742..5758)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5755..5777)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5766..5782
                     /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(5790..5796,5797..5814,5815..5820))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5835..5856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5973..5990)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6144..6732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6224..6243)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6903..7694,7695..7763))
                     /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     7526..7545
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7764..7865,1..3))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcgcacattt ccccgaaaag tgccacctga cgtcgacaaa aaacgagaga ccggggcgtc
       61 gaaccccgct ggctaggttt tagagacccg ctggtcgccg gaccgtcccc cggtgtttcg
      121 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
      181 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
      241 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttctaattat
      301 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
      361 ttcctgcccc tcgacaaaaa acgagagacc ggggcgtcga accccgctgg ctaggtttta
      421 gagacccgct ggtcgccgga ccgtcccccg gtgtttcgtc ctttccacaa gatatataaa
      481 gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat
      541 aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact
      601 aatatctttg tgtttacagt caaattaatt ctaattatct ctctaacagc cttgtatcgt
      661 atatgcaaat atgaaggaat catgggaaat aggccctctt cctgcccctc gacaaaaaac
      721 gagagaccgg ggcgtcgaac cccgctggct aggttttaga gacccgctgg tcgccggacc
      781 gtcccccggt gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc
      841 aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc
      901 caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca
      961 aattaattct aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca
     1021 tgggaaatag gccctcttcc tgcccctcga caaaaaacga gagaccgggg cgtcgaaccc
     1081 cgctggctag gttttagaga cccgctggtc gccggaccgt cccccggtgt ttcgtccttt
     1141 ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata
     1201 gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg
     1261 tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattctaa ttatctctct
     1321 aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctcttcctg
     1381 cccctcgagt ctagacccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     1441 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     1501 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     1561 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
     1621 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     1681 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     1741 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     1801 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     1861 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     1921 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     1981 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     2041 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     2101 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     2161 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
     2221 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgc
     2281 caccatgacc gtgaagtaca ccgacgccca gatccaacgc ctgcgcgagt atggtaatgg
     2341 cacctatgaa cagaaagttt ttgaggacct ggccagccgt gacgccgcgt tcagcaagga
     2401 aatgagcgtg gccagcaccg acaacgagaa gaaaatcaag ggtatgattg ccaatccgag
     2461 ccgccacggc ctgacccagc tgatgaacga catcgctgat gccctggtgg cggagggttt
     2521 cattgaagtg cgtaccccga tctttattag caaggatgcg ctggctcgca tgaccatcac
     2581 cgaagacaag ccgctgttca aacaggtgtt ttggattgat gagaaacgtg ccctgcgccc
     2641 gatgctggcg ccaaacctgc tgagcgtggc gcgtgacctg cgcgatcaca ccgacggtcc
     2701 ggtgaaaatc ttcgaaatgg gcagctgctt tcgtaaagag agccacagcg gkawryrmsy
     2761 gkggccagca ccgacaacga gaagaaaatc aagggtatga ttgccaatcc gagccgccac
     2821 ggcctgaccc agctgatgaa cgacatcgct gatgccctgg tggcggaggg tttcattgaa
     2881 gtgcgtaccc cgatctttat tagcaaggat gcgctggctc gcatgaccat caccgaagac
     2941 aagccgctgt tcaaacaggt gttttggatt gatgagaaac gtgccctgcg cccgatgctg
     3001 gcgccaaacc tgctgagcgt ggcgcgtgac ctgcgcgatc acaccgacgg tccggtgaaa
     3061 atcttcgaaa tgggcagctg ctttcgtaaa gagagccaca gcggtatgca cctggaggag
     3121 ttcaccatgc tgaatctgtt tgatatgggt ccgcgcggcg acgctaccga agtgctgaag
     3181 aactacatta gcgtggtgat gaaagctgcc ggcctgccgg actacgatct ggtgcaggaa
     3241 gagagcgacg tgtataagga aaccatcgac gtggaaatta acggtcagga agtgtgcagc
     3301 gcggctgtgg gtccacacta tctggatgcg gcccatgacg tgcatgagcc gtggagcggt
     3361 gctggttttg gcctggaacg tctgctgacc atccgcgaga aatactccac cgtgaaaaag
     3421 ggcggcgcaa gcatctccta cctgaatggt gccaaaatca attaagcggc cgctttaaac
     3481 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3541 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3601 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3661 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3721 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3781 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3841 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3901 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3961 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4021 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4081 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4141 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     4201 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     4261 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     4321 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4381 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4441 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     4501 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4561 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4621 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4681 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4741 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4801 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4861 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4921 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4981 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     5041 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     5101 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     5161 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     5221 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     5281 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     5341 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5401 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5461 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5521 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5581 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5641 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5701 tctgtatacc ggtcctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5761 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5821 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5881 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5941 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     6001 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6061 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6121 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6181 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6241 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6301 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6361 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6421 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6481 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6541 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6601 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6661 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6721 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6781 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6841 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6901 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6961 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     7021 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7081 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7141 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7201 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7261 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7321 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7381 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7441 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7501 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7561 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7621 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7681 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7741 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7801 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7861 gttcc
//
