LOCUS       sequence-367519-        7261 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mitochondria surface expression of HaloTag7 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7261)
  TITLE     Johnsson xHTLs
REFERENCE   2  (bases 1 to 7261)
  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-HaloTag7_T2A_mEGFP
FEATURES             Location/Qualifiers
     source          1..7261
                     /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
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     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             join(2456..2458,2459..3172)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2456..2458,2459..3169)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2498..2519)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2759..2778)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3106..3127
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3213..3230)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3219..3443
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3576..3595)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3727..3774
                     /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             3782..4804
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3782..4804
                     /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    4934..5055
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4971..4990)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5025..5044
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5104..5120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5104..5120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5117..5139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5128..5144
                     /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(5152..5158,5159..5176,5177..5182))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5197..5218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5335..5352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5506..6094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5586..5605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6265..7056,7057..7125))
                     /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     6888..6907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7126..7230)
                     /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 attcacgact 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 gcccagtgag
     2461 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2521 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2581 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2641 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2701 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2761 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2821 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2881 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2941 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     3001 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3061 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3121 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt gaccgctcga
     3181 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3241 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3301 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3361 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3421 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3481 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3541 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3601 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3661 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3721 tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc
     3781 caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     3841 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     3901 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     3961 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     4021 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     4081 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     4141 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     4201 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     4261 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     4321 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     4381 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     4441 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     4501 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     4561 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     4621 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     4681 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     4741 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     4801 atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4861 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4921 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4981 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     5041 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     5101 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5161 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5221 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5281 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5341 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5401 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5461 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5521 cccccctgac gagcatcaca aaaatcgacg ctcaagtcgg aggtggcgaa acccgacagg
     5581 actataaaga taccaggcgt ttccccctgg aagctccctc atgcgctctc ctgttccgac
     5641 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5701 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5761 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5821 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5881 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5941 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6001 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6061 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6121 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6181 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6241 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6301 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6361 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6421 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6481 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6541 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6601 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6661 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6721 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6781 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6841 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6901 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6961 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7021 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7081 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7141 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7201 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7261 c
//
