LOCUS       sequence-456408-        7175 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the expression of the passenger domain of the Orientia 
            tsutsugamushi protein ScaE in HeLa Flp-In cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7175)
  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 7175)
  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-pScaE
FEATURES             Location/Qualifiers
     source          1..7175
                     /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             join(987..989,990..992,993..1703)
                     /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     complement(1032..1053)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1293..1312)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(3127..3144)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3133..3357
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3490..3509)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3641..3688
                     /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             3696..4718
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3696..4718
                     /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    4848..4969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4885..4904)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4939..4958
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5018..5034)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5018..5034)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5031..5053)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5042..5058
                     /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(5066..5072,5073..5090,5091..5096))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5111..5132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5249..5266)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5420..6008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5500..5519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6179..6970,6971..7039))
                     /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     6802..6821
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7040..7144)
                     /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 cttaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     1021 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1081 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1141 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1201 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1261 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1321 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1381 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1441 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1501 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1561 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1621 ccaacgagaa gcgcgatcac atggtcctta aggagttcgt gaccgccgcc gggatcactc
     1681 tcggcatgga cgagctgtac aagggaggtt caggtggatc tggaggtacc aagcaggacc
     1741 agctgaacaa caagatcaac cagatcgatg ctagaaagtt tgagctggtc cagcaacaaa
     1801 gaaacctgga aagagataga gagaagatct ataatagaat cgacctggaa aagatcaaaa
     1861 gccacagcga catcctggat aagttcctgc tgcatccttc taatcaagtg accgtgtacc
     1921 tgaatgagga agatccccac gataggaaga agaacacaag aacccggagc agaatgctgt
     1981 acaaacacat ccaggatacc aagaaactga tcgacaaaat gcggcctagc atcgtggaca
     2041 tcgtcagcag ctacaccaac aacggccctg atgcccctga ccccgccaga ctcatcacca
     2101 tgaccgacca ggaggccaag ttcaacgaca acgacagagt ggcccgcttc gacgacagaa
     2161 tccacggcgt gaacaatccc ggcaagaagt ggcgggccgt gaccaccgag ataaacgaac
     2221 tgagccagga acttgacaag gtgttccagt tccaccaggc cctggtggac gtgcagaacc
     2281 agatcaaggc cctgtccctg cctagcctga atgacctggc caccgaggtg aagaaactgc
     2341 acctgggcat cggcgagcag ctcgaagata tcgacgagtc tatcaaggcc tgcgagcagg
     2401 gctctaatag catgctgaag ctgggacagt acgagcagct gctggatgac ctgagtacaa
     2461 catctaagaa catcctgcag atccttcaga agcacgtgca ggccggcaac gctcctgcca
     2521 caaaaaacga gctggaccag atgtacaagc agatgcagga tattaagacc ctgatgccta
     2581 agtttaccca ggacctggaa atgctgtcca aacagctgca cgacatcctg ggactgatcg
     2641 caaagctcga tcccagagcc gatgagctgg acaccacaaa acaccaggac cctggactga
     2701 gcgagcggat ccggaagatc gtgaacgaca tcatcaacga cttccctcca gtgctgcagc
     2761 aatacaaaaa caacctggaa aacgacctga aaattatcaa cgataagatc gctaatatcg
     2821 accagctggt ttccagcctg aaccagaagc tggacaccct gcagcaggag caactgctgt
     2881 ctgatcaaat cgtgccaaca aaggctatca catctttcga gaagcagcag cctctgcatc
     2941 aggccgctca gcacaacgcc atgctggcca acgccatcag cttcaccgcc gacaccgccg
     3001 tgtccgccac aaccagcatc ctgagcaaga tcgccacagt gcagaatcag agccccaaga
     3061 tcatcagctc aggcagcaac atctacaaca gcaacggctc cggcagctaa agcggccgct
     3121 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3181 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3241 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3301 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     3361 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     3421 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3481 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3541 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3601 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     3661 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     3721 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     3781 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     3841 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     3901 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     3961 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     4021 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     4081 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     4141 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     4201 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     4261 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     4321 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     4381 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     4441 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     4501 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     4561 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     4621 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     4681 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     4741 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4801 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4861 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4921 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4981 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5041 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5101 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5161 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5221 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5281 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5341 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5401 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5461 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5521 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5581 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5641 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5701 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5761 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5821 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5881 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5941 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6001 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6061 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctat atccttttaa
     6121 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6181 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6241 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6301 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6361 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6421 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6481 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6541 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6601 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6661 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6721 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6781 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6841 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6901 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6961 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7021 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7081 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7141 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
