LOCUS       sequence-464223-        8186 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Make PiggyBac stable cell line expressing Oropouche virus BeAn 19991
            Gc spike region (residues 482–894 of M polyprotein) with C-terminal 
            His6 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8186)
  TITLE     Serological assessment of the immune response during Oropouche virus
            infection in Brazil
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8186)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: PB OROV Gc Spike H6
FEATURES             Location/Qualifiers
     source          1..8186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(28..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     583..605
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     597..614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     621..640
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        621..639
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   706..768
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1140..1158
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1177..1195
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1214..1232
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1251..1269
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1288..1306
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1325..1343
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1362..1380
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1399..1417
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1439..1463
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1562..1571
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2861..2878
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    2912..3500
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2965..2985)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3383..3394)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3594..3611)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3600..3824
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4108..4155
                     /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)."
     rep_origin      4249..4383
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4388..4990
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4887..4907
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5224..5345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5261..5280)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5813..5847
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(5996..6014)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6035..6051)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6035..6051)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6048..6070)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6059..6075
                     /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(6083..6089,6090..6107,6108..6113))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6128..6149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6266..6283)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6437..7025)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6517..6536)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7196..7987,7988..8056))
                     /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     7819..7838
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8057..8161)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgaattg taatacgact cactataggg cgaattggag ctcggtattc
      661 acgacagcag gctgaataat aaaaaaatta gaaactatta tttaacccta gaaagataat
      721 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
      781 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
      841 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
      901 aacaaaaact caaaatttct tctataaagt aacaaaactt ttaaacattc tctcttttac
      961 aaaaataaac ttattttgta ctttaaaaac agtcatgttg tattataaaa taagtaatta
     1021 gcttaaccta tacataatag aaacaaatta tacttattag tcagtcagaa acaactttgg
     1081 cacatatcaa tattatgctc tcgttaatta actcgaggga tccaagctgg ccgcacgtct
     1141 ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat agagaaggta
     1201 cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc tccctatcag
     1261 tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc gacacgtctc
     1321 gagctcccta tcagtgatag agaaggtacg tctagaacgt ctccctatca gtgatagaga
     1381 agtcgacacg tctcgagctc cctatcagtg atagagaagg taccccctat ataagcagag
     1441 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
     1501 gaagacaccg ggaccgatcc agcctccgcg gtggcggcca tcacaagttt gtacagctag
     1561 cgccaccatg gccaacctga tcatcatcag catggtgctg ggcgtcgcct acggcggatc
     1621 cgatgaggac tgcctgagca aggacatcaa gatcacctac caagagctgc acaactgcat
     1681 cggccccaag atcatgggcg atacctgcgt gtccaagagc gagctgtact ccgacctgtt
     1741 cagcaagaac ctggtcaccg agtacgacaa gaagtacttt gagcccgaca ccgtgaacga
     1801 ccagttcaac aagatcgagt tcgcccagga cgcccaccgg atgatcctgc tggaaagaat
     1861 cctgtacaag accgagtgcg agatgctgag cctgaagaag aacagcggcc cctacaatgt
     1921 ggcctggcgg acctacttca agaaccacaa catcgacctg tgcagccggc acaactacaa
     1981 gatgatctgc cagtgcatca acacccacag catgtgcaag aacaccgaca tcgactacaa
     2041 caaagagatc gaaacctact acaagagcaa cgccgctgcc tacagagccg acctgaacac
     2101 catcatggac accctgaaaa ccgccttccg gggcctgaca aaggtgctga tcgagaacta
     2161 catcgagaag gacgacagcg acgccctgaa ggccctgttc tccaacatca ccgacagcgt
     2221 gcaggacaat taccagatga tcggcatcct gaagttcgcc agcaagctgc tggacatcaa
     2281 cctgggcaga agcaccagaa gcgcccacca cagcatcatg accaacgaga tccccaagag
     2341 caatcccttc accgactaca gctacagcaa cctgaacatc aaagaatgca tgagccccga
     2401 gtctctgaag tgcttcaaga agcgggacag cacccctcac accaaccatc tgctgtgcaa
     2461 gatcgacaac aagtacaagg ccttcgactg gcccgagatc gagacaatcc agaaaggcca
     2521 gaagctgtgc ctgggcgaca gccactgcaa ccttgagttt accgccatca ccgccgataa
     2581 gatcatgagc ctgaccaact gctacaaaga gagcttcaca gcccagcctg ccgatatgca
     2641 ggccggcatc aagaagtgta gcgccgacga gatcggcgag tgcaccacac tggaagataa
     2701 gacctggcct atcatcttct gcggcggcaa gtactactac agcgactcca aagagcacgc
     2761 caaggacggc agcatcaaca actactgtct gaccaacaag tgcagcgagc agcggttccc
     2821 catccacgag aactggttta agaagtgcaa ctgggacaag catcaccacc atcatcactg
     2881 acttaagggt accgaattcg atatcaagct taatcaacct ctggattaca aaatttgtga
     2941 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     3001 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     3061 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     3121 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3181 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3241 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3301 ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     3361 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     3421 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     3481 cctttgggcc gcctccccgc gcggccgcat agataactga tccagtgtgc tgggctacga
     3541 tgcatgggcc ccctaggtcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     3601 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3661 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3721 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3781 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3841 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3901 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3961 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4021 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4081 tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga
     4141 aagtatagga acttccttgg ccaaaaagac cggtaccagg caggcagaag tatgcaaagc
     4201 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4261 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4321 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4381 ggctaccatg ggtaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     4441 ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     4501 cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     4561 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     4621 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     4681 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     4741 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     4801 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     4861 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     4921 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     4981 cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca
     5041 tggctccgac cgaagccacc cggggcggcc ccgccgaccc cgcacccgcc cccgaggccc
     5101 accgactcta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     5161 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     5221 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5281 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5341 tcttaacgct cgagtgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5401 tgtctgtata ccgtcgacct ctagctgcag gaattcgata tcaagctcta gccaacaagc
     5461 tcgtcatcgc tttgcagaag agcagagagg atatgctcat cgtctaaaga actacccatt
     5521 ttattatata ttagtcacga tatctataac aagaaaatat atatataata agttatcacg
     5581 taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta
     5641 aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact
     5701 taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg
     5761 cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc
     5821 aattttacgc agactatctt tctagggtta aaaaagattt gcgctttact cgacctaaac
     5881 tttaaacacg tcatagaatc ttcgtttgac aaaaaccaca ttgtggccaa gctgtgtgac
     5941 gcgacgcgcg ctaaagaatg gcaaaccaag tcgcgcgagg taccagcttt tgttcccttt
     6001 agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     6061 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     6121 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     6181 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6241 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6301 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6361 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6421 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6481 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6541 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6601 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6661 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6721 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6781 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6841 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6901 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6961 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7021 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7081 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7141 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7201 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7261 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7321 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7381 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7441 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7501 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7561 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7621 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7681 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7741 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7801 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7861 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7921 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7981 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     8041 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     8101 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     8161 gcacatttcc ccgaaaagtg ccacct
//
