LOCUS       sequence-359609-        6143 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of membrane protein with N-terminal FLAG tag and 
            C-terminal split GFP1-10 fragment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6143)
  AUTHORS   Bruguera ES, Mahoney JP, Weis WI
  TITLE     Reconstitution of purified membrane protein dimers in lipid 
            nanodiscs with defined stoichiometry and orientation using a split 
            GFP tether.
  JOURNAL   J Biol Chem. 2022 Jan 21:101628. doi: 10.1016/j.jbc.2022.101628.
  PUBMED    35074428
REFERENCE   2  (bases 1 to 6143)
  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: "J Biol 
            Chem. 2022 Jan 21:101628. doi: 10.1016/j.jbc.2022.101628."
COMMENT     Sequence Label: FLAG-3C-sGFP1-10_pcDNA3.1(-)
FEATURES             Location/Qualifiers
     source          1..6143
                     /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"
     CDS             790..813
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(853..881)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(1500..1517)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1506..1730
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1776..2204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1863..1882)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2073..2094
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2213..2233)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2218..2547
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2398..2533
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2460..2479
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2614..3408
                     /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(2668..2687)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3278..3297
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3582..3703
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3619..3638)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3673..3692
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3752..3768)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3752..3768)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3765..3787)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3776..3792
                     /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(3800..3806,3807..3824,3825..3830))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3845..3866
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3983..4000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4154..4742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4234..4253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4913..5704,5705..5773))
                     /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     5536..5555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5774..5878)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5953..5972)
                     /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 gctagcgttt aaacgggccc tctagactcg agcggccgcg ccaccatgaa gacgatcatc
      721 gccctgagct acatcttctg cctggtgttc gccgactaca aggacgatga tgacgccacc
      781 ggtggatccc tcgaagtcct tttccagggc cctggtggca ctagtagcaa aggagaagaa
      841 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa
      901 ttttctgtca gaggagaggg tgaaggtgat gctacaatcg gaaaactcac ccttaaattt
      961 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tctgacctat
     1021 ggtgttcaat gcttttcccg ttatccggat cacatgaaaa ggcatgactt tttcaagagt
     1081 gccatgcccg aaggttatgt acaggaacgc actatatctt tcaaagatga cgggaaatac
     1141 aagacgcgtg ctgtagtcaa gtttgaaggt gatacccttg ttaatcgtat cgagttaaag
     1201 ggtactgatt ttaaagaaga tggaaacatt ctcggacaca aactcgagta caactttaac
     1261 tcacacaatg tatacatcac ggcagacaaa caaaagaatg gaatcaaagc taacttcaca
     1321 gttcgccaca acgttgaaga tggttccgtt caactagcag accattatca acaaaatact
     1381 ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtcgac acaaactgtc
     1441 ctttcgaaag atcccaacga aaagtgaggt accaagctta agtttaaacc gctgatcagc
     1501 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     1561 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     1621 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     1681 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     1741 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     1801 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     1861 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     1921 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     1981 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2041 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2101 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2161 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2221 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2281 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2341 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     2401 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     2461 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     2521 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     2581 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     2641 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     2701 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     2761 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     2821 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     2881 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     2941 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3001 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3061 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3121 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3181 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3241 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3301 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3361 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     3421 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     3481 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     3541 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     3601 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3661 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     3721 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3781 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3841 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3901 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3961 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4021 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4081 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4141 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4201 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4261 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4321 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4381 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4441 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4501 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4561 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4621 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4681 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4741 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4801 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4861 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4921 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4981 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5041 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5101 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5161 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5221 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5281 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5341 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5401 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5461 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5521 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5581 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5641 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5701 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5761 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5821 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5881 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     5941 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6001 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     6061 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     6121 tgtacgggcc agatatacgc gtt
//
