LOCUS       sequence-376955-        7510 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Co-expression of dHaloTag7 at the mitochondria surface and HaloTag7 
            on actin filaments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7510)
  TITLE     Johnsson xHTLs
REFERENCE   2  (bases 1 to 7510)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pCDNA5/FRT/TO_TOM20-dHaloTag7_T2A_Lifeact-Halo...
FEATURES             Location/Qualifiers
     source          1..7510
                     /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             1508..2392
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHA
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             2402..2455
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2456..2506
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             2534..3418
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(3462..3479)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3468..3692
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3825..3844)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3976..4023
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4031..5053
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4031..5053
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5183..5304
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5220..5239)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5274..5293
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5353..5369)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5353..5369)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5366..5388)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5377..5393
                     /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(5401..5407,5408..5425,5426..5431))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5446..5467
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5584..5601)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5755..6343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5835..5854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6514..7305,7306..7374))
                     /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     7137..7156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7375..7479)
                     /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 cactagtcca gtgtggtgga
     1021 attctgcaga tatccagcaa ccatggtggg tcggaacagc gccatcgccg ccggtgtatg
     1081 cggggccctt ttcattgggt actgcatcta cttcgaccgc aaaagacgaa gtgaccccaa
     1141 cttcaagaac aggcttcgag aacgaagaaa gaaacagaag cttgccaagg agagagctgg
     1201 gctttccaag ttacctgacc ttaaagatgc tgaagctgtt cagaagttct tccttgaaga
     1261 aatacagctt ggtgaagagt tactagctca aggtgaatat gagaagggcg tagaccatct
     1321 gacaaatgca attgctgtgt gtggacagcc acagcagtta ctgcaggtct tacagcaaac
     1381 tcttccacca ccagtgttcc agatgcttct gactaagctc ccaacaatta gtcagagaat
     1441 tgtaagtgct cagagcttgg ctgaagatga tgtggaaggc ggtagcgggg atccaccggt
     1501 cgccaccgaa atcggtactg gctttccatt cgacccccat tatgtggaag tcctgggcga
     1561 gcgcatgcac tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg
     1621 taacccgacc tcctcctacg tgtggcgcaa catcatcccg catgttgcac cgacccatcg
     1681 ctgcattgct ccagacctga tcggtatggg caaatccgac aaaccagacc tgggttattt
     1741 cttcgacgac cacgtccgct tcatggatgc cttcatcgaa gccctgggtc tggaagaggt
     1801 cgtcctggtc attcacgcct ggggctccgc tctgggtttc cactgggcca agcgcaatcc
     1861 agagcgcgtc aaaggtattg catttatgga gttcatccgc cctatcccga cctgggacga
     1921 atggccagaa tttgcccgcg agaccttcca ggccttccgc accaccgacg tcggccgcaa
     1981 gctgatcatc gatcagaacg tttttatcga gggtacgctg ccgatgggtg tcgtccgccc
     2041 gctgactgaa gtcgagatgg accattaccg cgagccgttc ctgaatcctg ttgaccgcga
     2101 gccactgtgg cgcttcccaa acgagctgcc aatcgccggt gagccagcga acatcgtcgc
     2161 gctggtcgaa gaatacatgg actggctgca ccagtcccct gtcccgaagc tgctgttctg
     2221 gggcacccca ggcgttctga tcccaccggc cgaagccgct cgcctggcca aaagcctgcc
     2281 taactgcaag gctgtggaca tcggcccggg tctgaatctg ctgcaagaag acaacccgga
     2341 cctgatcggc agcgagatcg cgcgctggct gtccacgctc gagatttccg gcggcagtgg
     2401 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaatggg
     2461 cgtggccgac ctgatcaaga agttcgagag catcagcaag gaggaggggg atccaccggt
     2521 cgccaccgga tccgaaatcg gtactggctt tccattcgac ccccattatg tggaagtcct
     2581 gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct
     2641 gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac
     2701 ccatcgctgc attgctccag acctgatcgg tatgggcaaa tccgacaaac cagacctggg
     2761 ttatttcttc gacgaccacg tccgcttcat ggatgccttc atcgaagccc tgggtctgga
     2821 agaggtcgtc ctggtcattc acgactgggg ctccgctctg ggtttccact gggccaagcg
     2881 caatccagag cgcgtcaaag gtattgcatt tatggagttc atccgcccta tcccgacctg
     2941 ggacgaatgg ccagaatttg cccgcgagac cttccaggcc ttccgcacca ccgacgtcgg
     3001 ccgcaagctg atcatcgatc agaacgtttt tatcgagggt acgctgccga tgggtgtcgt
     3061 ccgcccgctg actgaagtcg agatggacca ttaccgcgag ccgttcctga atcctgttga
     3121 ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc gccggtgagc cagcgaacat
     3181 cgtcgcgctg gtcgaagaat acatggactg gctgcaccag tcccctgtcc cgaagctgct
     3241 gttctggggc accccaggcg ttctgatccc accggccgaa gccgctcgcc tggccaaaag
     3301 cctgcctaac tgcaaggctg tggacatcgg cccgggtctg aatctgctgc aagaagacaa
     3361 cccggacctg atcggcagcg agatcgcgcg ctggctgtct actctggaga tttccggtta
     3421 accgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     3481 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3541 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3601 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3661 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     3721 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3781 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3841 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3901 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3961 tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac
     4021 ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa
     4081 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag
     4141 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta
     4201 caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct
     4261 tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt
     4321 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc
     4381 catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc
     4441 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca
     4501 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct
     4561 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga
     4621 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag
     4681 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg
     4741 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg
     4801 atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt
     4861 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg
     4921 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac
     4981 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag
     5041 ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct atgaaaggtt
     5101 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     5161 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     5221 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5281 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     5341 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     5401 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5461 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5521 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5581 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5641 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5701 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5761 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcgga ggtggcgaaa
     5821 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctca tgcgctctcc
     5881 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5941 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6001 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6061 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6121 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6181 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6241 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6301 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6361 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6421 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6481 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6541 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6601 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6661 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6721 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6781 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6841 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6901 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6961 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7021 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7081 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7141 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7201 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7261 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7321 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7381 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7441 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7501 acctgacgtc
//
