LOCUS       sequence-127354-        6747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of The CFP-tagged GIV-CT biosensors .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6747)
  AUTHORS   Midde KK, Aznar N, Laederich MB, Ma GS, Kunkel MT, Newton AC, Ghosh 
            P
  TITLE     Multimodular biosensors reveal a novel platform for activation of G 
            proteins by growth factor receptors.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):E937-46. doi: 
            10.1073/pnas.1420140112. Epub 2015 Feb 17.
  PUBMED    25713130
REFERENCE   2  (bases 1 to 6747)
  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: 
            "10.1073/pnas.1420140112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-03-3- 3"; volume: "112"; issue: "9"; pages: "E937-46"
COMMENT     Sequence Label: CFP-GIV-CT WT
FEATURES             Location/Qualifiers
     source          1..6747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(923..925,926..928,929..1639)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(968..989)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1229..1248)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1576..1597
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2341..2358)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2347..2571
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2617..3045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2704..2723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2914..2935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3054..3074)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3059..3388
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3239..3374
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3301..3320
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3455..4249
                     /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(3509..3528)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4119..4138
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4423..4544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4460..4479)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4514..4533
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4593..4609)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4593..4609)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4606..4628)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4617..4633
                     /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(4641..4647,4648..4665,4666..4671))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4686..4707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4824..4841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4995..5580)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5075..5094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5751..6542,6543..6611))
                     /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     6374..6393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6612..6716)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccatggtgag caagggcgag gagctgttca ccggggtggt
      961 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aggttcagcg tgtccggcga
     1021 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1081 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tggggcgtgc agtgcttcag
     1141 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1201 cgtccaggag cgtaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1261 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1321 ggacggcaac atcctggggc acaagctgga gtacaactac atcagccaca acgtctatat
     1381 caccgccgac aagcagaaga acggcatcaa ggcccacttc aagatccgcc acaacatcga
     1441 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1501 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     1561 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1621 catggacgag ctgtacaagg gaggctcagg gatccatggc tctgaaacac tggagagtcg
     1681 acatcacaag atcaaaactg gttcccctgg aagtgaagtt gttactctac aacagttttt
     1741 ggaagaaagc aataagctta cctcagtaca gataaagtcc tcaagtcaag agaatctttt
     1801 agatgaagta atgaaaagtt tgtctgtctc ttctgacttt ttgggaaaag acaaaccagt
     1861 tagctgtggt ctggccaggt cagtaagtgg aaaaacccca ggggacttct atgatagacg
     1921 gacaactaag cctgagtttt tgagacctgg tcctcgaaaa actgaagata cctacttcat
     1981 tagttctgcg ggaaaaccta caccaggcac tcaaggaaaa ataaaattag taaaagaatc
     2041 ttctctgtca cgacaatcaa aagatagtaa cccttatgca actttacctc gtgcaagcag
     2101 cgtgatctca actgccgaag gaactacacg aaggacaagc atccatgatt ttttgaccaa
     2161 ggacagtaga ctgcctatat cagttgattc accaccagct gctgctgaca gcaacaccac
     2221 tgcagcatct aatgtggaca aagtacaaga aagcagaaat tcaaaaagca ggtctaggga
     2281 gcaacaaagc tcctaagcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag
     2341 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2401 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2461 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2521 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     2581 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     2641 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2701 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2761 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2821 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2881 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2941 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3001 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     3061 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3121 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3181 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3241 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     3301 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     3361 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     3421 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3481 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3541 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3601 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     3661 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3721 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3781 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3841 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3901 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3961 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4021 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4081 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4141 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4201 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4261 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4321 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4381 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     4441 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4501 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4561 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4621 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4681 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4741 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4801 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4861 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4921 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4981 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5041 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5101 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5161 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5221 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5281 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5341 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5401 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5461 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5521 gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5581 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5641 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5701 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5761 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5821 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5881 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5941 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6001 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6061 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6121 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6181 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6241 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6301 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6361 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6421 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6481 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6541 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6601 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6661 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6721 tttccccgaa aagtgccacc tgacgtc
//
