LOCUS       sequence-310521-        8053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the translational pair that enables efficient 
            incorporation of multiple phenylalanine-based amino acids in 
            response to the amber codon into proteins in mammmalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8053)
  AUTHORS   Bottke T, Ernicke S, Serfling R, Ihling C, Burda E, Gurevich VV, 
            Sinz A, Coin I
  TITLE     Exploring GPCR-arrestin interfaces with genetically encoded 
            crosslinkers.
  JOURNAL   EMBO Rep. 2020 Sep 14:e50437. doi: 10.15252/embr.202050437.
  PUBMED    32929862
REFERENCE   2  (bases 1 to 8053)
  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"; journalName: "EMBO Rep. 
            2020 Sep 14:e50437. doi: 10.15252/embr.202050437."
COMMENT     Sequence Label: MmXYRS-4xM15
FEATURES             Location/Qualifiers
     source          1..8053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2064..2081)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2070..2294
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2340..2768
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2427..2446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2637..2658
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2777..2797)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2782..3111
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2962..3097
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3024..3043
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3178..3972
                     /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(3232..3251)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3842..3861
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4146..4267
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4183..4202)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4237..4256
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4314..4330)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4314..4330)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4327..4349)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4338..4354
                     /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(4362..4368,4369..4386,4387..4392))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4407..4428
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4545..4562)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4716..5304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4796..4815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5475..6266,6267..6335))
                     /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     6098..6117
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6336..6440)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6550..6798)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(6889..7137)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(7228..7476)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(7567..7815)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7863..7882)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgcca ccatggacaa gaagcccctg aacaccctga tcagcgccac cggcctgtgg
      721 atgagcagaa ccggcaccat ccacaagatc aagcaccacg aggtgtcccg gtccaagatc
      781 tacatcgaga tggcctgcgg cgaccacctg gtcgtgaaca acagcagaag cagccggacc
      841 gccagagccc tgagacacca caagtacaga aagacctgca agcggtgccg ggtgtccgac
      901 gaggacctga acaagttcct gaccaaggcc aacgaggacc agaccagcgt gaaagtgaag
      961 gtggtgtccg cccccacccg gaccaagaaa gccatgccta agagcgtggc cagggccccc
     1021 aagcccctgg aaaacacaga agccgctcag gcccagccca gcggcagcaa attttctcct
     1081 gccatccccg tgtccaccca ggaatccgtg tctgtgcctg cctccgtgtc taccagcatc
     1141 agcagcatct ctaccggcgc cacagccagc gctctcgtga agggcaacac caaccccatc
     1201 accagcatga gcgcccctgt gcaggcttct gcccctgccc tgacaaagag ccagaccgac
     1261 agactggaag tgctgctgaa ccccaaggac gagatcagcc tgaacagcgg caagcctttc
     1321 agagagctgg aaagcgagct gctgagccgg cggaagaagg acctgcagca gatctacgcc
     1381 gaggaaagag agaactacct gggcaagctg gaacgggaaa tcacccggtt cttcgtggac
     1441 cggggcttcc tggaaatcaa gagccccatc ctgatccccc tggagtacat cgagcggatg
     1501 ggcatcgaca acgacaccga gctgagcaag cagatcttca gggtggacaa aaacttctgc
     1561 ctgcggccca tgctgacccc caacctgtac aactatctgc ggaagctgga tagagccctg
     1621 cccgacccca tcaagatctt cgagatcggc ccctgctaca gaaaagagag cgacggcaaa
     1681 gagcacctgg aagagttcac catgctggcc ttcgcccaga tgggcagcgg ctgcaccaga
     1741 gagaacctgg aatctatcat caccgacttt ctgaaccacc tggggatcga cttcaagatc
     1801 gtgggcgaca gctgcatggt gttcggcgac accctggacg tgatgcacgg cgacctggaa
     1861 ctgtctagcg ccgtcgtggg acccatccct ctggacagag agtggggcat cgataagccc
     1921 accatcggag ccggcttcgg cctggaaaga ctgctgaaag tgaaacacga tttcaagaac
     1981 atcaagaggg ccgccagaag cgagagctac tacaacggca tcagcaccaa cctgtgagcg
     2041 gccgctttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2101 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2161 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2221 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2281 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2341 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2401 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2461 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2521 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2581 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2641 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2701 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2761 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2821 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2881 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2941 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3001 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3061 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3121 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     3181 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3241 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3301 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     3361 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3421 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     3481 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     3541 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     3601 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     3661 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     3721 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     3781 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     3841 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     3901 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     3961 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4021 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4081 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4141 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4201 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4261 tatcttatca tgtctgtata ccggtcctct agctagagct tggcgtaatc atggtcatag
     4321 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4381 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4441 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4501 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4561 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4621 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4681 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4741 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4801 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4861 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4921 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4981 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5041 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5101 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5161 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5221 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5281 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5341 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5401 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5461 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5521 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5581 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5641 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5701 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5761 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5821 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5881 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5941 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6001 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6061 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6121 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6181 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6241 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6301 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6361 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6421 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacaaaaaa
     6481 cggaaacccc gggaatcgaa cccggctgaa cggatttaga gtccgttcgg tctccctgac
     6541 caggtttccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt
     6601 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta
     6661 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt
     6721 caaattaatt ctaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat
     6781 catgggaaat aggccctctt cctgcccctc gacaaaaaac ggaaaccccg ggaatcgaac
     6841 ccggctgaac ggatttagag tccgttcggt ctccctgacc aggtttccgg tgtttcgtcc
     6901 tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg
     6961 atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct
     7021 acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc taattatctc
     7081 tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcttc
     7141 ctgcccctcg acaaaaaacg gaaaccccgg gaatcgaacc cggctgaacg gatttagagt
     7201 ccgttcggtc tccctgacca ggtttccggt gtttcgtcct ttccacaaga tatataaagc
     7261 caagaaatcg aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa
     7321 ttttaaaact gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa
     7381 tatctttgtg tttacagtca aattaattct aattatctct ctaacagcct tgtatcgtat
     7441 atgcaaatat gaaggaatca tgggaaatag gccctcttcc tgcccctcga caaaaaacgg
     7501 aaaccccggg aatcgaaccc ggctgaacgg atttagagtc cgttcggtct ccctgaccag
     7561 gtttccggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca
     7621 agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc
     7681 aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa
     7741 attaattcta attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat
     7801 gggaaatagg ccctcttcct gcccctcgag tctagacccg atcccctatg gtgcactctc
     7861 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     7921 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     7981 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     8041 agatatacgc gtt
//
