LOCUS       sequence-171121-        8654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pEZT-BM vector with alpha4 gene after CMV promoter, published 
            crystallization construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8654)
  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 8654)
  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 a4 nicotinic subunit crystallization con...
FEATURES             Location/Qualifiers
     source          1..8654
                     /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"
     misc_feature    3001..3589
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3054..3074)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3472..3483)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3731..3865
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3768..3787)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3822..3841
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  3894..4059
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      4243..4697
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4330..4349)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4539..4560
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4723..4827
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4828..4896,4897..5688)
                     /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(5046..5065)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5859..6447
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6348..6367
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6601..6618
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  6752..6976
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(7043..7576)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7765..7770,7771..7787,7788..7793))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    8301..8349
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     8374..8393
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(join(8434..8654,1..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(8476..8497)
                     /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 tggagctagg gggccccgga gcgccgcggc tgctgccgcc gctgctgctg
     1801 cttctgggga ccggcctcct gcgcgccagc agccatgtgg agacccgggc ccacgccgag
     1861 gagcggctcc tgaagaaact cttctccggt tacaacaagt ggtcccgacc cgtggccaac
     1921 atctcggacg tggtcctcgt ccgcttcggc ctgtccatcg ctcagctcat tgacgtggat
     1981 gagaagaacc agatgatgac cacgaacgta tgggtgaagc aggagtggca cgactacaag
     2041 ctgcgctggg acccagctga ctatgagaat gtcacctcca tccgcatccc ctccgagctc
     2101 atctggcggc cggacatcgt cctctacaac aatgctgacg gggacttcgc ggtcacccac
     2161 ctgaccaagg cccacctgtt ccatgacggg cgggtgcagt ggactccccc ggccatttac
     2221 aagagctcct gcagcatcga cgtcaccttc ttccccttcg accagcagaa ctgcaccatg
     2281 aaattcggct cctggaccta cgacaaggcc aagatcgacc tggtgaacat gcacagccgc
     2341 gtggaccagc tggacttctg ggagagtggc gagtgggtca tcgtggacgc cgtgggcacc
     2401 tacaacacca ggaagtacga gtgctgcgcc gagatctacc cggacatcac ctatgccttc
     2461 gtcatccggc ggctgccgct cttctacacc atcaacctca tcatcccctg cctgctcatc
     2521 tcctgcctca ccgtgctggt cttctacctg ccctccgagt gtggcgagaa gatcacgctg
     2581 tgcatctccg tgctgctgtc gctcaccgtc ttcctgctgc tcatcaccga gatcatcccg
     2641 tccacctcac tggtcatccc actcatcggc gagtacctgc tgttcaccat gatcttcgtc
     2701 accctgtcca tcgtcatcac ggtcttcgtg ctcaacgtgc accaccgctc gccacgcacg
     2761 cacaccatgc ccacctgggt acgcagggtc ttcctggaca tcgtgccacg actgctcctc
     2821 atgaagcggc cgtccgtggt cgacacagac ttcgaaagat cggtgaagga ggactggaag
     2881 tacgtggcca tggtcatcga ccgcatcttc ctctggatgt tcatcatcgt ctgcctgctg
     2941 gggacggtgg gcctctttct gccgccctgg ctggctggca tgatctaagg taccaagctt
     3001 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3061 ccttttacgc tatgtgaata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3121 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3181 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3241 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3301 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3361 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
     3421 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3481 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3541 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tgcagtctga
     3601 caagcttgtc gagaagtact agaggatcat aatcagccat accacatttg tagaggtttt
     3661 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     3721 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3781 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3841 caatgtatct tatcatgtct ggatctgatc actgcttgag cctaggagat ccgaaccaga
     3901 taagtgaaat ctagttccaa actattttgt catttttaat tttcgtatta gcttacgacg
     3961 ctacacccag ttcccatcta ttttgtcact cttccctaaa taatccttaa aaactccatt
     4021 tccacccctc ccagttccca actattttgt ccgcccacag cggggcattt ttcttcctgt
     4081 tatgttttta atcaaacatc ctgccaactc catgtgacaa accgtcatct tcggctactt
     4141 tttctctgtc acagaatgaa aatttttctg tcatctcttc gttattaatg tttgtaattg
     4201 actgaatatc aacgcttatt tgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     4261 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4321 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4381 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4441 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4501 tttttcgccc tttgacgttg gagtccacgt tcttaatagt ggactcttgt tccaaactgg
     4561 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4621 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4681 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4741 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4801 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4861 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4921 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4981 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5041 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5101 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5161 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5221 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5281 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5341 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5401 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5461 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5521 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5581 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5641 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5701 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5761 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5821 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5881 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5941 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6001 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6061 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6121 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6181 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6241 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6301 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6361 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6421 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6481 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6541 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6601 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     6661 ctgtgcggta tttcacaccg catagaccag ccgcgtaacc tggcaaaatc ggttacggtt
     6721 gagtaataaa tggatgccct gcgtaagcgg gtgtgggcgg acaataaagt cttaaactga
     6781 acaaaataga tctaaactat gacaataaag tcttaaacta gacagaatag ttgtaaactg
     6841 aaatcagtcc agttatgctg tgaaaaagca tactggactt ttgttatggc taaagcaaac
     6901 tcttcatttt ctgaagtgca aattgcccgt cgtattaaag aggggcgtgg ccaagggcat
     6961 ggtaaagact atattcgcgg cgttgtgaca atttaccgaa caactccgcg gccgggaagc
     7021 cgatctcggc ttgaacgaat tgttaggtgg cggtacttgg gtcgatatca aagtgcatca
     7081 cttcttcccg tatgcccaac tttgtataga gagccactgc gggatcgtca ccgtaatctg
     7141 cttgcacgta gatcacataa gcaccaagcg cgttggcctc atgcttgagg agattgatga
     7201 gcgcggtggc aatgccctgc ctccggtgct cgccggagac tgcgagatca tagatataga
     7261 tctcactacg cggctgctca aacttgggca gaacgtaagc cgcgagagcg ccaacaaccg
     7321 cttcttggtc gaaggcagca agcgcgatga atgtcttact acggagcaag ttcccgaggt
     7381 aatcggagtc cggctgatgt tgggagtagg tggctacgtc tccgaactca cgaccgaaaa
     7441 gatcaagagc agcccgcatg gatttgactt ggtcagggcc gagcctacat gtgcgaatga
     7501 tgcccatact tgagccacct aactttgttt tagggcgact gccctgctgc gtaacatcgt
     7561 tgctgctgcg taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc
     7621 gcttgctgct tggatgcccg aggcatagac tgtacaaaaa aacagtcata acaagccatg
     7681 aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt
     7741 gagcgcatac gctacttgca ttacagttta cgaaccgaac aggcttatgt caactgggtt
     7801 cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc
     7861 gaggcatttc tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag
     7921 gcattggcgg ccttgctgtt cttctacggc aaggtgctgt gcacggatct gcccttgctt
     7981 caggagatcg gtagacctcg gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa
     8041 gtggttcgca tcctcggttt tctggaaggc gagcatcgtt tgttcgccca ggactctagc
     8101 tatagttcta gtggttggct acgtacccgt agtggctatg gcagggcttg ccgccccgac
     8161 gttggctgcg agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga
     8221 aacgcgggcg tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg
     8281 ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt
     8341 ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc
     8401 tcccccatct cccggttccg catgctatgc atccttgtac agctcgtcca tgccgagagt
     8461 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     8521 tttgctcagc ttggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc
     8581 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt
     8641 gtggcggatc ttga
//
