LOCUS       sequence-476290-        6872 bp    DNA     circular SYN 18-JUN-2025
DEFINITION  A fluorescence reporter to show incorporation of target unnatural 
            amino acids..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6872)
  TITLE     Genetic code expansion reveals site-specific lactylation in living 
            cells reshapes protein functions
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6872)
  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            "Unpublished"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: pcDNA-mCherry-TAG-EGFP
FEATURES             Location/Qualifiers
     source          1..6872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..30)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        202..581
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        582..785
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     736..756
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     830..849
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        830..848
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             902..1615
                     /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
                     EGRHSTGGMDELYKG"
     primer_bind     complement(1047..1065)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1315..1334
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(1625..1627,1628..2338)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2339..2356
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2430..2447)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2436..2660
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2706..3134
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2793..2812)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3003..3024
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3143..3163)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3148..3477
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3328..3463
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3390..3409
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3544..4338
                     /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(3598..3617)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4208..4227
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4512..4633
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4549..4568)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4603..4622
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4682..4698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4682..4698)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4695..4717)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4706..4722
                     /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(4730..4736,4737..4754,4755..4760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4775..4796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4913..4930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5084..5672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5164..5183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5843..6634,6635..6703))
                     /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     6466..6485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6704..6808)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
       61 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
      121 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      181 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      241 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      301 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      361 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      421 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      481 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      541 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      601 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      661 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      721 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      781 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
      841 actataggga gacccaagct ggctagcgtt taaacttaag cttggtaccg agctcggatc
      901 catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
      961 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     1021 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     1081 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     1141 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     1201 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     1261 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     1321 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     1381 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     1441 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     1501 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     1561 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg gttagggtgg
     1621 tagcgtgagc aaaggcgaag aactgttcac cggtgtggtg ccgattctgg ttgaactgga
     1681 cggcgatgtg aacggccaca aattcagcgt gagtggcgaa ggtgaaggcg atgcgaccta
     1741 cggcaaactg acgctgaagt tcatctgcac cacgggcaaa ctgccggttc catggccgac
     1801 gctggtgacc acgctgacct atggcgtgca atgcttcagt cgctacccgg accacatgaa
     1861 gcagcacgac ttcttcaaaa gcgcgatgcc agaaggctac gtgcaagaac gcaccatctt
     1921 cttcaaggac gacggcaact acaaaacccg cgccgaggtg aaattcgagg gtgatacgct
     1981 ggtgaaccgc atcgagctca agggcatcga cttcaaagaa gacggcaaca ttctgggcca
     2041 caagctggag tataactaca acagccacaa cgtgtacatc atggcggaca agcagaaaaa
     2101 cggcatcaaa gttaacttca agatccgcca caacatcgag gatggcagcg tgcagctggc
     2161 cgatcattac cagcagaata cgccgattgg cgacggtccg gttctgctgc cagataacca
     2221 ctatctgagc acgcagagcg cgctgagcaa agatccgaac gagaagcgcg atcacatggt
     2281 tctgctggaa ttcgtgaccg ccgcgggcat caccctcggt atggatgaac tgtacaaaca
     2341 tcatcatcat catcattaag aattctgcag atatccagca cagtggcggc cgctcgagtc
     2401 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2461 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2521 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2581 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2641 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2701 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2761 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2821 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2881 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2941 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3001 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3061 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3121 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3181 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3241 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3301 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3361 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     3421 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3481 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     3541 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3601 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3661 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     3721 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     3781 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     3841 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     3901 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     3961 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4021 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     4081 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     4141 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4201 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4261 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4321 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4381 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4441 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4501 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4561 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4621 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4681 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4741 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4801 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4861 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4921 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4981 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5041 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5101 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5161 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5221 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5281 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5341 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5401 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5461 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5521 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5581 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5641 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5701 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5761 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5821 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5881 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5941 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6001 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6061 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6121 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6181 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6241 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6301 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6361 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6421 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6481 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6541 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6601 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6661 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6721 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6781 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     6841 acggatcggg agatctcccg atcccctatg gt
//
