LOCUS        pIRE4-Azi        6658 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Plasmid encoding for the translational pair that enables efficent 
            incoporation of p-Azido-phenylalanine 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 6658)
  AUTHORS   Seidel L, Zarzycka B, Zaidi SA, Katritch V, Coin I
  TITLE     Structural insight into the activation of a class B 
            G-protein-coupled receptor by peptide hormones in live human cells.
  JOURNAL   Elife. 2017 Aug 3;6. doi: 10.7554/eLife.27711.
  PUBMED    28771403
REFERENCE   2  (bases 1 to 6658)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2017-08-3"; volume: "6. doi"; pages: " 10.7554/eLife.27711"
COMMENT     Sequence Label: pIRE4-Azi
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..525
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(61..84)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     458..477
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      552..561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1981..2102
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2018..2037)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2072..2091
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2127..2375
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        2485..2733
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        2843..3091
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        3201..3449
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     rep_origin      complement(3552..4007)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3689..3710)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3901..3920
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4034..4138
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4140..4497
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4140..4331
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4163..4183)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4348..4483
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4532..5326
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4532..5326
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4586..4605)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5196..5215
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5514..5533)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5558..5605
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5956..6544
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6445..6464
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgcgttagt tattaaaatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga
       61 gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc
      121 acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg
      181 ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc
      241 aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct
      301 gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct
      361 ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg
      421 ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc
      481 acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctg ctagcgttta
      541 aacttaagct tgccaccatg gccagcagca acctgatcaa gcagctgcag gaacggggcc
      601 tggtggccca agtgacagat gaagaagccc tggccgagag actggcccag ggacctattg
      661 ccctgctgtg cggctttgac cccaccgccg attctctgca cctgggacat ctggtgcctc
      721 tgctgtgtct gaagcggttc cagcaggccg gccacaaacc tgtggctctc gtgggcggag
      781 ccaccggcct gattggcgat cctagcttta aggccgccga gcggaagctg aacaccgaag
      841 agacagtgca ggaatgggtg gacaagatcc ggaaacaggt ggcccccttc ctggacttcg
      901 actgcggcga gaatagcgcc attgccgcca acaactacga ttggttcggc aatatgaacg
      961 tgctgacctt cctgcgggac atcggcaagc acttcagcgt gaaccagatg atcaacaaag
     1021 aagccgtgaa gcagcggctg aacagagagg accagggcat cagcttcacc gagttcagct
     1081 acaacctgct gcagggctac agcatggcct gcgccaacaa gcagtacggc gtggtgctgc
     1141 agatcggcgg ctctgaccag tggggcaata tcaccagcgg catcgacctg accagacggc
     1201 tgcaccagaa ccaggtgttc ggcctgaccg tgcccctgat cacaaaggcc gacggcacca
     1261 agttcggcaa gaccgaaggc ggagccgtgt ggctggaccc caagaaaacc agcccctaca
     1321 agttctacca gttctggatc aacaccgcca gagccgacgt gtaccggttc ctgaagttct
     1381 tcaccttcat gagcatcgag gaaatcaacg ccctggaaga agaggacaag aacagcggca
     1441 aggcccccag agcccagtac gtgctggcag aacaagtgac caggctggtg cacggcgagg
     1501 aaggactgca ggccgccaag agaatcaccg agtgcctgtt cagcggcagc ctgagcgctc
     1561 tgagcgaggc cgattttgag cagctggctc aggacggcgt gcccatggtg gaaatggaaa
     1621 agggcgccga cctgatgcag gccctggtgg attctgagct gcagcccagc agaggacagg
     1681 ccagaaagac aatcgccagc aacgccatca ccatcaacgg cgagaagcag agcgaccccg
     1741 agtacttctt caaagaggaa gatcggctgt tcggccggtt caccctgctg cggagaggca
     1801 agaagaacta ctgcctgatc tgctggaagt aatgaattct gcagatatcc agcacagtgg
     1861 cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta
     1921 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     1981 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2041 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2101 taaggtctag actcgagggg caggaagagg gcctatttcc catgattcct tcatatttgc
     2161 atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga
     2221 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2281 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2341 tggctttata tatcttgtgg aaaggacgaa acaccggagg ggtagcgaag tggctaaacg
     2401 cggcggactc taaatccgct ccctttgggt tcggcggttc gaatccgtcc ccctccagtc
     2461 ctttttttgg tcgaggggca ggaagagggc ctatttccca tgattccttc atatttgcat
     2521 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     2581 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2641 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2701 gctttatata tcttgtggaa aggacgaaac accggagggg tagcgaagtg gctaaacgcg
     2761 gcggactcta aatccgctcc ctttgggttc ggcggttcga atccgtcccc ctccagtcct
     2821 ttttttggtc gaggggcagg aagagggcct atttcccatg attccttcat atttgcatat
     2881 acgatacaag gctgttagag agataattag aattaatttg actgtaaaca caaagatatt
     2941 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     3001 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     3061 tttatatatc ttgtggaaag gacgaaacac cggaggggta gcgaagtggc taaacgcggc
     3121 ggactctaaa tccgctccct ttgggttcgg cggttcgaat ccgtccccct ccagtccttt
     3181 ttttggtcga ggggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac
     3241 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
     3301 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     3361 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     3421 tatatatctt gtggaaagga cgaaacaccg gaggggtagc gaagtggcta aacgcggcgg
     3481 actctaaatc cgctcccttt gggttcggcg gttcgaatcc gtccccctcc agtccttttt
     3541 ttggtcgacg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3601 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3661 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     3721 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     3781 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     3841 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     3901 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     3961 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     4021 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4081 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     4141 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     4201 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     4261 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4321 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4381 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4441 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     4501 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4561 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4621 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4681 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     4741 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4801 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4861 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4921 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4981 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     5041 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     5101 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     5161 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     5221 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     5281 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     5341 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     5401 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5461 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     5521 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     5581 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     5641 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     5701 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     5761 cctgccatag cagtactaaa tatttatccc gggctcaggt tactcatata tactttagat
     5821 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5881 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5941 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6001 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6061 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6121 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6181 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6241 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6301 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6361 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6421 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6481 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6541 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6601 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//