LOCUS       sequence-456404-        6458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the expression of the passenger domain of the Orientia 
            tsutsugamushi protein ScaC C-terminally fused to a mitochondrial 
            targeting sequence in HeLa Flp-In cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6458)
  AUTHORS   Manigrasso G, Saharat K, Kullapanich C, Boulanger J, Morgan TE, 
            Kramer H, Salje J, Carter AP
  TITLE     The intracellular bacterium Orientia tsutsugamushi hijacks the 
            adaptor protein BICD2 for dynein-based motility
  JOURNAL   bioRxiv 2024.04.11.589026
REFERENCE   2  (bases 1 to 6458)
  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: "bioRxiv 
            2024.04.11.589026"
COMMENT     Sequence Label: pcDNA5/FRT/TO_eGFP-pScaC-MTS
FEATURES             Location/Qualifiers
     source          1..6458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     80..97
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    214..235
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(250..255,256..273,274..280)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    288..304
                     /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)."
     primer_bind     293..315
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     312..328
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     312..328
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    377..498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(388..407)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     442..461
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(628..1650)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(628..1650)
                     /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
                     "
     protein_bind    complement(1658..1705)
                     /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)."
     primer_bind     1837..1856
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1989..2213
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2202..2219
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(2926..3636,3637..3639,3640..3642))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     3317..3336
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3576..3597
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3736..3760)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3770..3788
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3791..3809
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(3811..4014)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3840..3860)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4015..4394
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4566..4585
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4660..4764
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4765..4833,4834..5625)
                     /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(4983..5002)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5796..6384
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6285..6304
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
       61 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
      121 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
      181 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
      241 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
      301 acaatttcac acaggaaaca gctatgacca tgattacgcc aagctctagc tagaggtcga
      361 cggtatacag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
      421 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
      481 agctgcaata aacaagttgg ggtgggcgaa gaactccagc atgagatccc cgcgctggag
      541 gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc
      601 ggtggaatcg aaatctcgta gtacgtgcta ttcctttgcc ctcggacgag tgctggggcg
      661 tcggtttcca ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct
      721 gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg
      781 accctgcgcc caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc
      841 aagaccaatg cggagcatat acgcccggag ccgcggcgat cctgcaagct ccggatgcct
      901 ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat
      961 gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt
     1021 tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg
     1081 gacttcgggg cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc
     1141 actgacggtg tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca
     1201 tatgaaatca cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc
     1261 gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga ccggctgcag
     1321 aacagcgggc agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga
     1381 gatgcaatag gtcaggctct cgctgaattc cccaatgtca agcacttccg gaatcgggag
     1441 cgcggccgat gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct
     1501 atttacccgc aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc
     1561 gccctccgag agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc
     1621 gacagacgtc gcggtgagtt caggcttttt ggccaaggaa gttcctatac tttctagaga
     1681 ataggaactt cggaatagga acttctaggt acgtgaacca tcaccctaat caagtttttt
     1741 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     1801 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     1861 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     1921 taatgcgccg ctacagggcg cgtggggata ccccctagag ccccagctgg ttctttccgc
     1981 ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc
     2041 ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg
     2101 cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa
     2161 ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc
     2221 tgatcagcgg ccgctttagt tgttcttgcg cagctggatg atcttgatga aggcgcccag
     2281 gctgaacacg ccgatggcca gcatggccag gatcagagag ccgctgccgt tcagcttcac
     2341 gctgttgatc agggcggggt tgttgttctt gttctgcagg aacaggctat tctgcagggt
     2401 cacttgcagc tgctgcagct ggatcttcag ctgctcgttc tgctttctca ggtcgtcgat
     2461 ctcggctctt gtctctggca ggataggttg caggtcggtg ttcttggcca gcacctgttc
     2521 cagtttagct tccagctgct gcagcttgat tttaagctgg ttgtttttct gctgcaggta
     2581 gtcgttttcc ttgttcaggc tctcgatctc tgtaatcagc agtctgatgt aatcagactg
     2641 ctctgtagcc ctggtattag attgctgcag ccgcatggtc agattcttgc tgctggtggt
     2701 gctctgttcc agcatgcaca ggagcttgga cacttcggcg atgtccttcc gcagtccctc
     2761 tctggcctcg cccatattgc tgatgtccag gttcatattg gacaccacgt tgctcagatc
     2821 gttgtcggcg acctcttcct catcttcctt gtggatgttc tcggcctgag gacacttgcc
     2881 cttctcaggg gtgatgcttt tacctccaga tccacctgaa cctcccttgt acagctcgtc
     2941 catgccgaga gtgatcccgg cggcggtcac gaactcctta aggaccatgt gatcgcgctt
     3001 ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag
     3061 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc
     3121 gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc
     3181 catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc
     3241 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa
     3301 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg
     3361 gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta
     3421 gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg
     3481 cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc
     3541 gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat
     3601 gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtaagct taagtttaaa
     3661 cgctagagtc cggaggctgg atcggtcccg gtgtcttcta tggaggtcaa aacagcgtgg
     3721 atggcgtctc caggcgatct gacggttcac taaacgagct cgtcgacgat ctctatcact
     3781 gatagggaga tctctatcac tgatagggag agctctgctt atatagacct cccaccgtac
     3841 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     3901 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     3961 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     4021 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     4081 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     4141 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     4201 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     4261 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     4321 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt
     4381 caataatcaa tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct
     4441 aaccctaagc agattcttca tgcaattgtc ggtcaagcct tgccttgttg tagcttaaat
     4501 tttgctcgcg cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa
     4561 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aggggatcgg gagatctccc
     4621 gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4681 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4741 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4801 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4861 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4921 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4981 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5041 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5101 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5161 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5221 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5281 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5341 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5401 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5461 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5521 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5581 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5641 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatataggtg
     5701 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5761 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5821 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5881 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5941 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6001 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6061 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6121 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6181 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6241 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6301 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6361 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6421 ttttgctggc cttttgctca catgttcttt cctgcgtt
//
