LOCUS       sequence-171117-        8702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pEZT-BM vector with beta2 gene after CMV promoter, published 
            crystallization construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8702)
  AUTHORS   Morales-Perez CL, Noviello CM, Hibbs RE
  TITLE     X-ray structure of the human alpha4beta2 nicotinic receptor.
  JOURNAL   Nature. 2016 Oct 3;538(7625):411-415. doi: 10.1038/nature19785.
  PUBMED    27698419
REFERENCE   2  (bases 1 to 8702)
  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"; journalName: 
            "Nature"; date: "2016-10-3- 3"; volume: "538"; issue: "7625"; pages:
            "411-415"
COMMENT     Sequence Label: Human b2 nicotinic subunit crystallization con...
FEATURES             Location/Qualifiers
     source          1..8702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     171..190
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     430..451
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(518..627)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     enhancer        812..1191
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1192..1395
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1346..1366
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1392..1416
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1531..1663
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1646..1665
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1708..1726
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3010..3033
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     misc_feature    3049..3637
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3102..3122)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3520..3531)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3779..3913
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3816..3835)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3870..3889
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  3942..4107
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      4291..4745
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4378..4397)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4587..4608
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4771..4875
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4876..4944,4945..5736)
                     /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     complement(5094..5113)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5907..6495
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6396..6415
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6649..6666
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  6800..7024
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(7091..7624)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7813..7818,7819..7835,7836..7841))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    8349..8397
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     8422..8441
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(join(8482..8702,1..490,491..493))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(8524..8545)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt
       61 agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca
      121 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt
      181 tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact
      241 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg
      301 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact
      361 tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt
      421 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
      481 cgcccttgct caccgggtga tcaagtcttc gtcgagtgat tgtaaataaa atgtaattta
      541 cagtatagta ttttaattaa tatacaaatg atttgataat aattcttatt taactataat
      601 atattgtgtt gggttgaatt aaaggtccgt atactccgga atattaatag gatcccggtc
      661 cgagcgcgcg gaattcaata ttggccatta gccatattat tcattggtta tatagcataa
      721 atcaatattg gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat
      781 attggctcat gtccaatatg accgccatgt tggcattgat tattgactag ttattaatag
      841 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      901 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      961 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     1021 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct
     1081 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg
     1141 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     1201 ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     1261 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     1321 tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     1381 tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt
     1441 tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc
     1501 tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa
     1561 ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca
     1621 cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc
     1681 aattacagct cttaaggcta gagtacttaa tacgactcac tataggctag cctcgaggcg
     1741 gccgccacca tggcccggcg ctgcggcccc gtggcgctgc tccttggctt cggcctcctc
     1801 cggctgtgct caggggtgtg gggtacggat acagaggagc ggctggtgga gcatctcctg
     1861 gatccttccc gctacaacaa gcttatccgc ccagccacca atggctctga gctggtgaca
     1921 gtacagctta tggtgtcact ggcccagctc atcagtgtgc atgagcggga gcagatcatg
     1981 accaccaatg tctggctgac ccaggagtgg gaagattatc gcctcacctg gaagcctgaa
     2041 gagtttgaca acatgaagaa agttcggctc ccttccaaac acatctggct cccagatgtg
     2101 gtcctgtaca acaatgctga cggcatgtac gaggtgtcct tctattccaa tgccgtggtc
     2161 tcctatgatg gcagcatctt ctggctgccg cctgccatct acaagagcgc atgcaagatt
     2221 gaagtaaagc acttcccatt tgaccagcag aactgcacca tgaagttccg ttcgtggacc
     2281 tacgaccgca cagagatcga cttggtgctg aagagtgagg tggccagcct ggacgacttc
     2341 acacctagtg gtgagtggga catcgtggcg ctgccgggcc ggcgcaacga gaaccccgac
     2401 gactctacgt acgtggacat cacgtatgac ttcatcattc gccgcaagcc gctcttctac
     2461 accatcaacc tcatcatccc ctgtgtgctc atcacctcgc tagccatcct tgtcttctac
     2521 ctgccatccg actgtggcga gaagatgacg ttgtgcatct cagtgctgct ggcgctcacg
     2581 gtcttcctgc tgctcatctc caagatcgtg cctcccacct ccctcgacgt gccgctcgtc
     2641 ggcaagtacc tcatgttcac catggtgctt gtcaccttct ccatcgtcac cagcgtgtgc
     2701 gtgctcaacg tgcaccaccg ctcgcccacc acgcacacca tggcgccctg ggtgaaggtc
     2761 gtcttcctgg agaagctgcc cgcgctgctc ttcatgcagc agccacgcca tcatgacgat
     2821 gaccaggaaa gaagcgtgag tgaggactgg aagtacgtcg ccatggtgat cgaccgcctc
     2881 ttcctctgga tctttgtctt tgtctgtgtc tttggcacca tcggcatgtt cctgcagcct
     2941 ctcttccaga actacaccac caccaccttc ctccactcag accactcagc ccccagctcc
     3001 aagtccgcct ggtcccaccc ccagttcgag aagtgaggta ccaagcttaa tcaacctctg
     3061 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3121 tgtgaatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3181 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3241 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3301 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3361 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     3421 aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc
     3481 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     3541 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     3601 cagacgagtc ggatctccct ttgggccgcc tccccgcctg cagtctgaca agcttgtcga
     3661 gaagtactag aggatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     3721 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     3781 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3841 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3901 tcatgtctgg atctgatcac tgcttgagcc taggagatcc gaaccagata agtgaaatct
     3961 agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct acacccagtt
     4021 cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc cacccctccc
     4081 agttcccaac tattttgtcc gcccacagcg gggcattttt cttcctgtta tgtttttaat
     4141 caaacatcct gccaactcca tgtgacaaac cgtcatcttc ggctactttt tctctgtcac
     4201 agaatgaaaa tttttctgtc atctcttcgt tattaatgtt tgtaattgac tgaatatcaa
     4261 cgcttatttg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     4321 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4381 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4441 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4501 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4561 tgacgttgga gtccacgttc ttaatagtgg actcttgttc caaactggaa caacactcaa
     4621 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4681 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     4741 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4801 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4861 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4921 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4981 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5041 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5101 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5161 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5221 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5281 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5341 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5401 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5461 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5521 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5581 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5641 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5701 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5761 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5821 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5881 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5941 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6001 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6061 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6121 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6181 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6241 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6301 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6361 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6421 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6481 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6541 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6601 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     6661 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     6721 tcacaccgca tagaccagcc gcgtaacctg gcaaaatcgg ttacggttga gtaataaatg
     6781 gatgccctgc gtaagcgggt gtgggcggac aataaagtct taaactgaac aaaatagatc
     6841 taaactatga caataaagtc ttaaactaga cagaatagtt gtaaactgaa atcagtccag
     6901 ttatgctgtg aaaaagcata ctggactttt gttatggcta aagcaaactc ttcattttct
     6961 gaagtgcaaa ttgcccgtcg tattaaagag gggcgtggcc aagggcatgg taaagactat
     7021 attcgcggcg ttgtgacaat ttaccgaaca actccgcggc cgggaagccg atctcggctt
     7081 gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact tcttcccgta
     7141 tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct tgcacgtaga
     7201 tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc gcggtggcaa
     7261 tgccctgcct ccggtgctcg ccggagactg cgagatcata gatatagatc tcactacgcg
     7321 gctgctcaaa cttgggcaga acgtaagccg cgagagcgcc aacaaccgct tcttggtcga
     7381 aggcagcaag cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa tcggagtccg
     7441 gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga tcaagagcag
     7501 cccgcatgga tttgacttgg tcagggccga gcctacatgt gcgaatgatg cccatacttg
     7561 agccacctaa ctttgtttta gggcgactgc cctgctgcgt aacatcgttg ctgctgcgta
     7621 acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg
     7681 gatgcccgag gcatagactg tacaaaaaaa cagtcataac aagccatgaa aaccgccact
     7741 gcgccgttac caccgctgcg ttcggtcaag gttctggacc agttgcgtga gcgcatacgc
     7801 tacttgcatt acagtttacg aaccgaacag gcttatgtca actgggttcg tgccttcatc
     7861 cgtttccacg gtgtgcgtca cccggcaacc ttgggcagca gcgaagtcga ggcatttctg
     7921 tcctggctgg cgaacgagcg caaggtttcg gtctccacgc atcgtcaggc attggcggcc
     7981 ttgctgttct tctacggcaa ggtgctgtgc acggatctgc ccttgcttca ggagatcggt
     8041 agacctcggc cgtcgcggcg cttgccggtg gtgctgaccc cggatgaagt ggttcgcatc
     8101 ctcggttttc tggaaggcga gcatcgtttg ttcgcccagg actctagcta tagttctagt
     8161 ggttggctac gtacccgtag tggctatggc agggcttgcc gccccgacgt tggctgcgag
     8221 ccctgggcct tcacccgaac ttgggggttg gggtggggaa aaggaagaaa cgcgggcgta
     8281 ttggtcccaa tggggtctcg gtggggtatc gacagagtgc cagccctggg accgaacccc
     8341 gcgtttatga acaaacgacc caacacccgt gcgttttatt ctgtcttttt attgccgtca
     8401 tagcgcgggt tccttccggt attgtctcct tccgtgtttc agttagcctc ccccatctcc
     8461 cggttccgca tgctatgcat ccttgtacag ctcgtccatg ccgagagtga tcccggcggc
     8521 ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagctt
     8581 ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt
     8641 gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt
     8701 ga
//
