LOCUS       sequence-183279-        7238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Methanosarcina barkeri pyrrolysyl-tRNA synthetase/tRNA pair 
            in mammalian cells to incorporate the photocrosslinkers DiZPK, 
            DiZSeK, or DiZHSeC into amber codon site on a protein of interest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7238)
  AUTHORS   Lin S, Yan H, Li L, Yang M, Peng B, Chen S, Li W, Chen PR
  TITLE     Site-specific engineering of chemical functionalities on the surface
            of live hepatitis D virus.
  JOURNAL   Angew Chem Int Ed Engl. 2013 Dec 23;52(52):13970-4. doi: 
            10.1002/anie.201305787. Epub 2013 Nov 12.
  PUBMED    24222614
REFERENCE   2  (bases 1 to 7238)
  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: 
            "10.1002/anie.201305787"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2013-12-23- 23"; volume: "52"; issue: "52"; pages: "13970-4"
COMMENT     Sequence Label: pCMV-MbPylRS(DiZPK)
FEATURES             Location/Qualifiers
     source          1..7238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..442
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        443..646
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     597..617
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     643..667
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        877..895
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     877..894
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     908..927
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      960..969
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2239..2256)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2245..2469
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2515..2943
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2602..2621)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2812..2833
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2952..2972)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2957..3286
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3137..3272
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3199..3218
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3335..4360
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3335..4360
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4490..4611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4527..4546)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4581..4600
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4660..4676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4660..4676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4673..4695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4684..4700
                     /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(4708..4714,4715..4732,4733..4738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4753..4774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4891..4908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5062..5650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5142..5161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5821..6612,6613..6681))
                     /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     6444..6463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6682..6786)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6876..7124)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(6934..6953)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7104..7124)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat gaccgccatg
       61 ttggcattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      121 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      181 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      241 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      301 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc
      361 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt
      421 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata
      481 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      541 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca
      601 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
      661 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg
      721 atccagcctc cgcggccggg aacggtgcat tggaacgcgg attccccgtg ccaagagtga
      781 cgtaagtacc gcctatagag tctataggcc cacccccttg gcttcgttag aacgcggcta
      841 caattaatac ataaccttat gtatcataca catacgattt aggtgacact atagaataac
      901 atccactttg cctttctctc cacaggtgtc cactcccagg tccaactgca cggaagcttg
      961 ccaccatgga taaaaaacca ttagatgttt taatatctgc gaccgggctc tggatgtcca
     1021 ggactggcac gctccacaaa atcaagcacc atgaggtctc aagaagtaaa atatacattg
     1081 aaatggcgtg tggagaccat cttgttgtga ataattccag gagttgtaga acagccagag
     1141 cattcagaca tcataagtac agaaaaacct gcaaacgatg tagggtttcg gacgaggata
     1201 tcaataattt tctcacaaga tcaaccgaaa gcaaaaacag tgtgaaagtt agggtagttt
     1261 ctgctccaaa ggtcaaaaaa gctatgccga aatcagtttc aagggctccg aagcctctgg
     1321 aaaattctgt ttctgcaaag gcatcaacga acacatccag atctgtacct tcgcctgcaa
     1381 aatcaactcc aaattcgtct gttcccgcat cggctcctgc tccttcactt acaagaagcc
     1441 agcttgatag ggttgaggct ctcttaagtc cagaggataa aatttctcta aatatggcaa
     1501 agcctttcag ggaacttgag cctgaacttg tgacaagaag aaaaaacgat tttcagcggc
     1561 tctataccaa tgatagagaa gactacctcg gtaaactcga acgtgatatt acgaaatttt
     1621 tcgtagaccg gggttttctg gagataaagt ctcctatcct tattccggcg gaatacgtgg
     1681 agagaatggg tattaataat gatactgaac tttcaaaaca gatcttccgg gtggataaaa
     1741 atctctgctt gaggccaatg cttgccccga ctctgtataa ctatgcgcga aaactcgata
     1801 ggattttacc aggcccaata aaaattttcg aagtcggacc ttgttaccgg aaagagtctg
     1861 acggcaaaga gcacctggaa gaatttacta tggtgaactt cagtcagatg ggttcgggat
     1921 gtactcggga aaatcttgaa gctctcatca aagagtttct ggactatctg gaaatcgact
     1981 tcgaaatcgt aggagattcc tgtatggtct ttggggatac tcttgatata atgcacgggg
     2041 acctggagct ttcttcggca gtcgtcgggc cagtttctct tgatagagaa tggggtattg
     2101 acaaaccatg gataggtgca ggttttggtc ttgaacgctt gctcaaggtt atgcacggct
     2161 ttaaaaacat taagagggca tcaaggtccg aatcttacta taatgggatt tcaaccaatc
     2221 tataaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2281 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2341 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2401 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     2461 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     2521 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2581 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2641 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2701 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2761 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2821 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2881 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2941 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3001 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3061 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3121 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3181 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     3241 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     3301 ttgtatatcc attttcggat ctgatcagca cgtgatgaaa aagcctgaac tcaccgcgac
     3361 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     3421 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     3481 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     3541 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     3601 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     3661 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     3721 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     3781 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     3841 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     3901 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     3961 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     4021 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     4081 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     4141 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     4201 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     4261 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     4321 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtgctac gagatttcga
     4381 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4441 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4501 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4561 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4621 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4681 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4741 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4801 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4861 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4921 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4981 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5041 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5101 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5161 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5221 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5281 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5341 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5401 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5461 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5521 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5581 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5641 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5701 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5761 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5821 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5881 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5941 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6001 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6061 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6121 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6181 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6241 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6301 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6361 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6421 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6481 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6541 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6601 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6661 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6721 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6781 tccgcgcaca tttccccgaa aaatggcgga aaccccggga atctaacccg gctgaacgga
     6841 tttagagtcc attcgatcta catgatcagg tttccggtgt ttcgtccttt ccacaagata
     6901 tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta
     6961 aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt
     7021 tgtactaata tctttgtgtt tacagtcaaa ttaattctaa ttatctctct aacagccttg
     7081 tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctcttcctg cccgaccttc
     7141 gcgatgtacg ggccagatat acgccaatat tggccattag ccatattatt cattggttat
     7201 atagcataaa tcaatattgg ctattggcca ttgcatac
//
