LOCUS       sequence-363834-        7776 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes G418 resistance and PhiC31-excisable hTFAM gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7776)
  AUTHORS   Kozhukhar N, Spadafora D, Rodriguez YAR, Alexeyev MF
  TITLE     A Method for In Situ Reverse Genetic Analysis of Proteins Involved 
            mtDNA Replication.
  JOURNAL   Cells. 2022 Jul 11;11(14). pii: cells11142168. doi: 
            10.3390/cells11142168.
  PUBMED    35883613
REFERENCE   2  (bases 1 to 7776)
  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: "Cells."; 
            date: "2022-07-11"; volume: "11(14). pii: cells11142168. doi"; 
            pages: " 10.3390/cells11142168"
COMMENT     Sequence Label: pMA5460
FEATURES             Location/Qualifiers
     source          1..7776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     regulatory      1157..1166
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1905..1921
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1906..1922
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1947..1980
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     misc_feature    2027..2600
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2195..2212)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2422..2441
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2603..3400
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2603..3400
                     /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="MAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2660..2679)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3270..3289
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    3469..4057
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3522..3542)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3940..3951)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4065..4082
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        4245..4652
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     primer_bind     complement(5394..5413)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5513..5535
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5573..5591)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5659..5763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5764..5832,5833..6624)
                     /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     complement(5982..6001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6795..7383
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7284..7303
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7537..7554
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc tagtgtttgc gcctgcgtct
      661 gtactagttg gctaactaga tctgtatctg gcggtcccgc ggaagaactg acgagttcgt
      721 attcccggcc gcagcccctg ggagacgtcc cagcggcctc gggggcccgt tttgtggccc
      781 attctgtatc agttaaccta cccgagtcgg actttttgga gctccgccac tgtccgaggg
      841 gtacgtggct ttgttggggg acgagagaca gagacacttc ccgcccccgt ctgaattttt
      901 gctttcggtt ttacgccgaa accgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg
      961 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagctcgaca aagttaagta
     1021 atagtccctc tctccaagct cacttacagg cggccgggct gcaggaatta ttcttactcc
     1081 acacaggcat agctacgccc ccaactgaga gaactcaaag gttaccccag ttggggcact
     1141 acacgcgatc gaattcgcca ccatggcgtt tctccgaagc atgtggggcg tgctgagtgc
     1201 cctgggaagg tctggagcag agctgtgcac cggctgtgga agtcgactgc gctccccctt
     1261 cagttttgtg tatttaccga ggtggttttc atctgtcttg gcaagttgtc caaagaaacc
     1321 tgtaagttct taccttcgat tttctaaaga acaactaccc atatttaaag ctcagaaccc
     1381 agatgcaaaa actacagaac taattagaag aattgcccag cgttggaggg aacttcctga
     1441 ttcaaagaaa aaaatatatc aagatgctta tagggcggag tggcaggtat ataaagaaga
     1501 gataagcaga tttaaagaac agctaactcc aagtcagatt atgtctttgg aaaaagaaat
     1561 catggacaaa catttaaaaa ggaaagctat gacaaaaaaa aaagagttaa cactgcttgg
     1621 aaaaccaaaa agacctcgtt cagcttataa cgtttatgta gctgaaagat tccaagaagc
     1681 taagggtgat tcaccgcagg aaaagctgaa gactgtaaag gaaaactgga aaaatctgtc
     1741 tgactctgaa aaggaattat atattcagca tgctaaagag gacgaaactc gttatcataa
     1801 tgaaatgaag tcttgggaag aacaaatgat tgaagttgga cgaaaggatc ttctacgtcg
     1861 cacaataaag aaacaacgaa aatatggtgc tgaggagtgt taactcgagg tcgacggtat
     1921 cgataagctt gatcgcgtgt ggagtacgcg cccggggagc ccaagggcac gccctggcac
     1981 ccgcaccgcc aagtacatcc caagcaataa ctgatccccc ggccgccccc tctccctccc
     2041 cccccccnta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     2101 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     2161 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     2221 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     2281 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     2341 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     2401 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     2461 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
     2521 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggtttcc
     2581 ctttgaaaaa cacgataata ccatggccat tgaacaagat ggattgcacg caggttctcc
     2641 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     2701 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     2761 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     2821 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     2881 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     2941 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3001 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3061 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3121 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccacg gcgatgcctg
     3181 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3241 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3301 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     3361 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gtccggactc agatccagcg
     3421 ctgataaccc agcctccacc acaaacaagg atgccggtac cggccgataa tcaacctctg
     3481 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3541 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3601 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3661 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3721 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3781 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     3841 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     3901 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     3961 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     4021 cagacgagtc ggatctccct ttgggccgcc tccccgcatc ggcccaccac caccaccacc
     4081 acatgtgaat gatccaagct tatcgatagg cctaggccta tcgataggcc taggcctatc
     4141 gataggccta acacgagcca tagataaaat aaaagatttt atttagtctc cagaaaaagg
     4201 ggggaatgaa agaccccacc tgtaggtttg gcaagctagc tgcagtaacg ccattttgca
     4261 aggcatggaa aaataccaaa ccaagaatag agaagttcag atcaagggcg ggtacatgaa
     4321 aatagctaac gttgggccaa acaggatatc tgcggtgagc agtttcggcc ccggcccggg
     4381 gccaagaaca gatggtcacc gcagtttcgg ccccggcccg aggccaagaa cagatggtcc
     4441 ccagatatgg cccaaccctc agcagtttct taagacccat cagatgtttc caggctcccc
     4501 caaggacctg aaatgaccct gcgccttatt tgaattaacc aatcagcctg cttctcgctt
     4561 ctgttcgcgc gcttctgctt cccgagctct ataaaagagc tcacaacccc tcactcggcg
     4621 cgccagtcct ccgacagact gagtcgcccg ggtacccgtg ttctcaataa accctcttgc
     4681 agttgcatcc gactcgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact
     4741 acccgtcagc gggggtcttt cagtaggatc tcgaccgatg cccttgagag ccttcaaccc
     4801 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     4861 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga
     4921 ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca
     4981 cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc
     5041 cattatcgcc ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg
     5101 aggctggatg gccttcccca ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc
     5161 gttgcaggcc atgctgtcca ggcaggtaga tgacgaccat cagggacagc ttcaaggatc
     5221 gctcgcggct cttaccagcc taacttcgat cattggaccg ctgatcgtca cggcgattta
     5281 tgccgcctcg gcgagcacat ggaacgggtt ggcatggatt gtaggcgcct gatgcggtat
     5341 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     5401 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     5461 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5521 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     5581 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     5641 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5701 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5761 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5821 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5881 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5941 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6001 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6061 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6121 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6181 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6241 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6301 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6361 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6421 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6481 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6541 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6601 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6661 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6721 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6781 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6841 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6901 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6961 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7021 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7081 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7141 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7201 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7261 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7321 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7381 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7441 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     7501 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     7561 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagg
     7621 gtcgaggggg ggcccgcgat tagtccaatt tgttaaagac aggatatcag tggtccaggc
     7681 tctagttttg actcaacaat atcaccagct gaagcctata gagtacgagc catagataga
     7741 ataaaagatt ttatttagtc tccagaaaaa gggggg
//
