LOCUS       sequence-456407-        7814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the expression of the passenger domain of the Orientia 
            tsutsugamushi protein ScaD in HeLa Flp-In cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7814)
  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 7814)
  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-pScaD
FEATURES             Location/Qualifiers
     source          1..7814
                     /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(3766..3783)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3772..3996
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4129..4148)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4280..4327
                     /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             4335..5357
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4335..5357
                     /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    5487..5608
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5524..5543)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5578..5597
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5657..5673)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5657..5673)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5670..5692)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5681..5697
                     /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(5705..5711,5712..5729,5730..5735))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5750..5771
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5888..5905)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6059..6647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6139..6158)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6818..7609,7610..7678))
                     /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     7441..7460
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7679..7783)
                     /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 tggaggtatg accaagtcct
     1741 ctacagccaa cttcaaggca caactgagcg aacgcctccg gatgcaccaa cagtcaccca
     1801 acgaaagcga cgacgaagac gacatggagg ctgatatcaa atcccagcag cagttcgctg
     1861 atttcaagaa gcatcagaac tctaacaaca agtacgtcag caagatcttc gcactggaaa
     1921 cactgatcac caaggttccc agaatccctg ataaggagaa caacaccgca cccaatcaga
     1981 acaacgatgc caccgacgat tgggatgatg ataacatttc aagcaaagac ctgaagtcct
     2041 ctcagaaacc actgaccttc aaggagagag caaagaagat cgagcagacc ctcggaccaa
     2101 tctttgcagc aaagtctaac accaatacaa agcaacccac cgacaacaac gttaagaaac
     2161 cagtggcagc cattcccaat cccaatacta ctcctatagc caccactcat tacaatgaca
     2221 acattagcag taaggacctc aattcctccc agaagaccct ggccttcaag gaaagggcta
     2281 agaagataga gcagactctc ggaccaatct ttgctgctaa gagcaacacc aatacaaagc
     2341 agccaacagg caacaacgtt aagcaacacg tcatcatgat tcccaacacc aataccacat
     2401 ctattgcaac aactcactcc aacaacaaca tctcctctaa ggatctgaac tcctcaaacg
     2461 aagccctgac tttcaaagaa agggctaaga gaattgaaca aaccctggga cccatcttcg
     2521 ctgccaagtc aaacacaaac actaagcagc ctacaggcaa caacgtgaag cagcacgtca
     2581 tcatgatacc caacaccaac actacaagta ttgccaccac ccattccaac aacaacatct
     2641 cttctaagga tctcaactct agcaatgaag ctctgacatt caaagagagg gctaagagaa
     2701 tcgagcaaac cctgggtccc atcttcgcag ctaagtccaa tacaaacacc aaacaaccta
     2761 catcaaacaa tgtcaagaac ccagtggtgg tgatcccaac accaaacaca acaagtatcg
     2821 ccaccaccaa tagtaaagat aacatctcaa gcaaggatct gaatagcagc cagaagacac
     2881 tcaccatcaa ggagaaggca aagaagatcg aacagactct cggtccaatc ttcgcagcca
     2941 aatccaatac caacacaaag cagccaacta acaacaatgt taagaagcct gtggctgtca
     3001 ttccctcacc caacaccacc agtattgcta ctaccaattc taaggataac atctcctcca
     3061 aagatctgaa ctctagccag aagacactga ccatcaaaga gaaagctaag aagatcgagc
     3121 aaacactggg acctatcttc gcagccaaga gcaacactaa cacaaagcag cccactgaca
     3181 acaatgtcaa gaaaccagtc acagccattc ctaagcataa cataacacct atcactactc
     3241 ctacccacca gacactcatc tgctcagatc agtctaatga cgaaatcgag gatatctgct
     3301 atgacgcaga ggatgttctg gactgctctg caaaccagcc atctctcctg cctcccagta
     3361 ccctcaacaa tagcgctgtc cagcctctca acaacaacaa ccagcaattc gacaactcag
     3421 aacagaacaa gactattgag tccgagatag aagatgtcct ggactgcagc gctaatcagc
     3481 caagtctgct cctccctagt actctcaaca attctgcagt gcagccactc aacaacaaca
     3541 accagcagtt tgacaactcc gagcagaaca agaccattga gagcgagatc aaggttaagg
     3601 cttacttcaa agaacaggag aaggcactcc tcaaggctgc ccaaacaaac gacatcgcta
     3661 tcacatctgc tatcactgcc gtgacagcca ccactgtctc agccataacc tcctccatct
     3721 ctaagatgtg tcagaaccat cgctgataaa gcggccgctg atcagcctcg actgtgcctt
     3781 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3841 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3901 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3961 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     4021 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4081 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4141 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4201 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4261 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     4321 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     4381 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     4441 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     4501 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     4561 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     4621 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     4681 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     4741 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     4801 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     4861 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     4921 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     4981 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     5041 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     5101 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     5161 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     5221 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     5281 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     5341 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     5401 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5461 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     5521 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5581 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     5641 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5701 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5761 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5821 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5881 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5941 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6001 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6061 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6121 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6181 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6241 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6301 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg atgcgcctta tccggtaact
     6361 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6421 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6481 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6541 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6601 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6661 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6721 tgagattatc aaaaaggatc ttcacctata tccttttaaa ttaaaaatga agttttaaat
     6781 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6841 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6901 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6961 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     7021 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     7081 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7141 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7201 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7261 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7321 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7381 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7441 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7501 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7561 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7621 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7681 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7741 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7801 tgccacctga cgtc
//
