LOCUS       sequence-490906-        6109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  anchoring mTFP1 to the mitochondrial membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6109)
  AUTHORS   Vigano MA, Bieli D, Schaefer JV, Jakob RP, Matsuda S, Maier T, 
            Pluckthun A, Affolter M
  TITLE     DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo
            protein manipulations.
  JOURNAL   Biol Open. 2018 Oct 29;7(11). pii: bio.036749. doi: 
            10.1242/bio.036749.
  PUBMED    30237292
REFERENCE   2  (bases 1 to 6109)
  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: "Biol 
            Open."; date: "2018-10-29"; volume: "7(11). pii: bio.036749. doi"; 
            pages: " 10.1242/bio.036749"
COMMENT     Sequence Label: pMITO-mTFP1
FEATURES             Location/Qualifiers
     source          1..6109
                     /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"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1212..1919
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLK
                     GDVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA
                     VARNSTDGMDELYK"
     CDS             1992..2048
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     primer_bind     complement(2117..2134)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2123..2347
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2393..2821
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2480..2499)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2690..2711
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2830..2850)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2835..3164
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3015..3150
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3077..3096
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3212..3259
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3278..3652
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3782..3903
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3819..3838)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3873..3892
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3952..3968)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3952..3968)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3965..3987)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3976..3992
                     /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(4000..4006,4007..4024,4025..4030))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4045..4066
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4183..4200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4354..4942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4434..4453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5113..5904,5905..5973))
                     /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     5736..5755
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5974..6078)
                     /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 ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cgccaccatg agtctgactt
     1021 ccagttccag cgtacgagtt gaatggatcg cagcagttac cattgctgct gggacagctg
     1081 caattggtta tctagcttac aaaagatttt atgttaaaga tcatcgaaat aaagctatga
     1141 taaaccttca catccagaaa gacaacccca agatagtaca tgctggaagc ttaccggtcg
     1201 ccgaattcgg tatggtgagc aagggcgagg agaccacaat gggcgtaatc aagcccgaca
     1261 tgaagatcaa gctgaagatg gagggcaacg tgaatggcca cgccttcgtg atcgagggcg
     1321 agggcgaggg caagccctac gacggcacca acaccatcaa cctggaggtg aaggagggag
     1381 cccccctgcc cttctcctac gacattctga ccaccgcgtt cgcctacggc aacagggcct
     1441 tcaccaagta ccccgacgac atccccaact acttcaagca gtccttcccc gagggctact
     1501 cttgggagcg caccatgacc ttcgaggaca agggcatcgt gaaggtgaag tccgacatct
     1561 ccatggagga ggactccttc atctacgaga tacacctcaa gggcgagaac ttccccccca
     1621 acggccccgt gatgcagaag aagaccaccg gctgggacgc ctccaccgag aggatgtacg
     1681 tgcgcgacgg cgtgctgaag ggcgacgtca agcacaagct gctgctggag ggcggcggcc
     1741 accaccgcgt tgacttcaag accatctaca gggccaagaa ggcggtgaag ctgcccgact
     1801 atcactttgt ggaccaccgc atcgagatcc tgaaccacga caaggactac aacaaggtga
     1861 ccgtttacga gagcgccgtg gcccgcaact ccaccgacgg catggacgag ctgtacaagg
     1921 gtggtggtgg cgcggccgca gatcgatcga tcacgcgtgc caccatgact agttttgtac
     1981 aaagcggtcc ggaagaactt ttgagcaaga attatcatct tgagaacgaa gtggctcgtc
     2041 ttaagaaaac cgcggtaaac atatggatcg atcataatac tcgagtctag agggcccgtt
     2101 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     2161 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     2221 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2281 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2341 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     2401 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2461 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2521 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2581 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2641 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2701 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2761 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2821 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2881 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     2941 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3001 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3061 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3121 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     3181 gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat
     3241 cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt
     3301 tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg
     3361 gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct
     3421 gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt
     3481 gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga
     3541 cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct
     3601 gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct
     3661 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     3721 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     3781 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3841 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3901 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     3961 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4021 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4081 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4141 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4201 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4261 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4321 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4381 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4441 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4501 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4561 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4621 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4681 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4741 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4801 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4861 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4921 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4981 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5041 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5101 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5161 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5221 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5281 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5341 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5401 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5461 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5521 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5581 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5641 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5701 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5761 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5821 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5881 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5941 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6001 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6061 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
