LOCUS       sequence-359608-        5552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of membrane protein with N-terminal FLAG tag and 
            C-terminal split GFP11 fragment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5552)
  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 5552)
  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-sGFP11_pcDNA3.1(-)
FEATURES             Location/Qualifiers
     source          1..5552
                     /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"
     CDS             826..873
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     primer_bind     complement(909..926)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    915..1139
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1185..1613
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1272..1291)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1482..1503
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1622..1642)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1627..1956
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1807..1942
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1869..1888
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2023..2817
                     /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(2077..2096)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2687..2706
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2991..3112
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3028..3047)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3082..3101
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3161..3177)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3161..3177)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3174..3196)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3185..3201
                     /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(3209..3215,3216..3233,3234..3239))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3254..3275
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3392..3409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3563..4151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3643..3662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4322..5113,5114..5182))
                     /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     4945..4964
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5183..5287)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5362..5381)
                     /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 cctggttctg gttctcgtga ccacatggtg
      841 ctgcatgaat atgtgaacgc ggcgggcatc acgtgaggta ccaagcttaa gtttaaaccg
      901 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
      961 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     1021 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     1081 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     1141 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     1201 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1261 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1321 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1381 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1441 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1501 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1561 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     1621 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     1681 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     1741 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     1801 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     1861 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     1921 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     1981 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     2041 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     2101 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     2161 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     2221 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     2281 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     2341 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     2401 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     2461 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     2521 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     2581 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     2641 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     2701 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     2761 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     2821 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     2881 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     2941 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     3001 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3061 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3121 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3181 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3241 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3301 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3361 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3421 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3481 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3541 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3601 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3661 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3721 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3781 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3841 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3901 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3961 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4021 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4081 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4141 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4201 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4261 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4321 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4381 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4441 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4501 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4561 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4621 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4681 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4741 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4801 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4861 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4921 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4981 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5041 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5101 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5161 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5221 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5281 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     5341 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     5401 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     5461 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     5521 gcttcgcgat gtacgggcca gatatacgcg tt
//
