LOCUS       sequence-399574-        6508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET donor: Calmodulin-2 tagged with N-terminal mTurquoise2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6508)
  AUTHORS   Feldmann C, Schanzler M, Ben-Johny M, Wahl-Schott C
  TITLE     Protocol for deriving proximity, affinity, and stoichiometry of 
            protein interactions using image-based quantitative two-hybrid FRET.
  JOURNAL   STAR Protoc. 2023 Jul 28;4(3):102459. doi: 
            10.1016/j.xpro.2023.102459.
  PUBMED    37516972
REFERENCE   2  (bases 1 to 6508)
  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: "STAR 
            Protoc."; date: "2023-07-28"; volume: "4(3):102459. doi"; pages: " 
            10.1016/j.xpro.2023.102459"
COMMENT     Sequence Label: mTq2-Calmodulin
FEATURES             Location/Qualifiers
     source          1..6508
                     /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(901..903,904..906,907..1617)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(946..967)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1207..1226)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1554..1575
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1624..2073
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPT
                     EAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAA
                     ELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     complement(2099..2116)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2105..2329
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2375..2803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2462..2481)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2672..2693
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2812..2832)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2817..3146
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2997..3132
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3059..3078
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3213..4007
                     /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(3267..3286)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3877..3896
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4181..4302
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4218..4237)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4272..4291
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4351..4367)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4351..4367)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4364..4386)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4375..4391
                     /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(4399..4405,4406..4423,4424..4429))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4444..4465
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4582..4599)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4753..5341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4833..4852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5512..6303,6304..6372))
                     /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     6135..6154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6373..6477)
                     /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 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      961 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1021 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1081 ctcgtgacca ccctgtcctg gggcgtgcag tgcttcgccc gctaccccga ccacatgaag
     1141 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1201 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1261 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1321 aagctggagt acaactactt tagcgacaac gtctatatca ccgccgacaa gcagaagaac
     1381 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc
     1441 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1501 tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1561 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggga
     1621 tccatggctg accaactgac tgaagagcag atcgcagaat tcaaagaagc tttctcccta
     1681 tttgacaagg acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct
     1741 ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac
     1801 ggtaatggca caatcgactt ccctgaattc ctgacaatga tggcaagaaa aatgaaagac
     1861 acagacagtg aagaagaaat tagagaagcg ttccgtgtgt ttgataagga tggcaatggc
     1921 tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat
     1981 gaagaggttg atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac
     2041 gaagagtttg tacaaatgat gacagcgaag tgagggcccg tttaaacccg ctgatcagcc
     2101 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2161 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2221 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2281 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     2341 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     2401 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2461 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2521 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2581 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2641 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2701 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2761 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     2821 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2881 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     2941 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3001 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3061 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     3121 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     3181 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3241 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3301 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3361 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     3421 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3481 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3541 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3601 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3661 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3721 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3781 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3841 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3901 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3961 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4021 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4081 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4141 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     4201 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4261 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     4321 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4381 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4441 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4501 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4561 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4621 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4681 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4741 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4801 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4861 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4921 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4981 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5041 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5101 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5161 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5221 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5281 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5341 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5401 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5461 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5521 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5581 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5641 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5701 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5761 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5821 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5881 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5941 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6001 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6061 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6121 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6181 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6241 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6301 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6361 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6421 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6481 atttccccga aaagtgccac ctgacgtc
//
