LOCUS       sequence-478445-        6739 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Mammalian expression of plasma membrane anchored (GAP43) 
            GRKct-Venus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6739)
  AUTHORS   Jang W, Senarath K, Feinberg G, Lu S, Lambert NA
  TITLE     Visualization of endogenous G proteins on endosomes and other 
            organelles.
  JOURNAL   Elife. 2024 Nov 8;13:RP97033. doi: 10.7554/eLife.97033.
  PUBMED    39514269
REFERENCE   2  (bases 1 to 6739)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2024-11-8"; volume: "13:RP97033. doi"; pages: " 
            10.7554/eLife.97033"
COMMENT     Sequence Label: memGRKct-Venus
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1335..2129)
                     /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(1446..1465)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2056..2075
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2196..2525)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2210..2345
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2264..2283)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2510..2530
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2539..2967)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2649..2670)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2861..2880
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3013..3237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3226..3243
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(3275..3988,3989..3991,3992..3994))
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3320..3341)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3669..3688
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3928..3949
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4691..4710)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4692..4710)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4755..4958)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4784..4804)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4959..5338
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5510..5529
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5604..5708
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5709..5777,5778..6569)
                     /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(5927..5946)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     1321 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     1381 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     1441 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     1501 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     1561 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     1621 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     1681 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     1741 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     1801 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     1861 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     1921 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     1981 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     2041 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     2101 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     2161 gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa
     2221 aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa
     2281 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     2341 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     2401 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     2461 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     2521 cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2581 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2641 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2701 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2761 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2821 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2881 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2941 cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt
     3001 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     3061 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     3121 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     3181 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     3241 aggctgatca gcgggtttaa acgggccctc tagattactt gtacagctcg tccatgccga
     3301 gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg
     3361 ggtctttgct cagcttggac tggtagctca ggtagtggtt gtcgggcagc agcacggggc
     3421 cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgccg ccgtcctcga
     3481 tgttgtggcg gatcttgaag ttggccttga tgccgttctt ctgcttgtcg gcggtgatat
     3541 agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct
     3601 tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct
     3661 cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc
     3721 cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcgggcga
     3781 agcactgcag gccgtagccg agggtggtca cgagggtggg ccagggcacg ggcagcttgc
     3841 cggtggtgca gatgagcttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc
     3901 cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca
     3961 ccccggtgaa cagctcctcg cccttgctca ccatggatcc acctccgagg ccgttgctgt
     4021 tcctatggca gaggggaggc tttgagagtt ccacgacggc tgagcgggat ttgttgagga
     4081 acttgggggc tcgccgtagc agccgctggg cctccatgaa tgtctccgtc agctctttct
     4141 tccactgcac aaactctggg tcactctcac actgcaaaac gaactgcttc cctcctttta
     4201 ttctcaacaa aatgcacttt ttgtctttaa tctgagtttc ttccacggac acaatctgtt
     4261 ccattgtcag taaactttgt cgcgactcgc cttctcctct ccactcaagt ctgttcggaa
     4321 agaggtaaaa atagcggcgc tgccactggg tcaggaaagg gttccccagc ttcagcatgt
     4381 acccgtgcac gatgcagtct cttcccaggg cgtaatcttc ttcgtggcca agctgcttat
     4441 ttttagctct cttcctggcc tcgattttat ccgtgtctgc atttactgct tcataaactg
     4501 tttccgccac ttcctgctgc cagcgctcag agatcaccag agggaagttc ttgtagagtt
     4561 cttggtcgca atcaagaagc ttgattccga tcttttggtc ctcatcattc ttttcaacct
     4621 gtttggttct tctcagacag cacagcatgg tggcgaccgg tggatcagat ctgctagcca
     4681 gcttgggtct ccctatagtg agtcgtatta atttcgataa gccagtaagc agtgggttct
     4741 ctagttagcc agagagctct gcttatatag acctcccacc gtacacgcct accgcccatt
     4801 tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa
     4861 aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc
     4921 tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat
     4981 acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag
     5041 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     5101 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
     5161 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     5221 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     5281 ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcaa
     5341 cgcgtatatc tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct aagcagattc
     5401 ttcatgcaat tgtcggtcaa gccttgcctt gttgtagctt aaattttgct cgcgcactac
     5461 tcagcgacct ccaacacaca agcagggagc agatactggc ttaactatgc ggcatcagag
     5521 cagattgtac tgagagtgca ccatagggga tcgggagatc tcccgatccg tcgacgtcag
     5581 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5641 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5701 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5761 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5821 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5881 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5941 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6001 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6061 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6121 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6181 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6241 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6301 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6361 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6421 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6481 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6541 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6601 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6661 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6721 aaaagatcaa aggatcttc
//
