LOCUS       62056_20975        7482 bp DNA     circular SYN 24-MAY-2021
DEFINITION  Cloning vector pTarget-eGFP-iFlp-neo, complete sequence.
ACCESSION   MW246026
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7482)
  AUTHORS   Dias MM, Vidigal J, Sequeira DP, Alves PM, Teixeira AP, Roldao A.
  TITLE     Insect High FiveTM cell line development using site-specific flipase
            recombination technology
  JOURNAL   G3 (Bethesda) (2021) In press
  PUBMED    33982066
REFERENCE   2  (bases 1 to 7482)
  AUTHORS   Dias MM, Vidigal J, Sequeira DP, Alves PM, Teixeira AP, Roldao A.
  TITLE     Direct Submission
  JOURNAL   Submitted (11-NOV-2020) Animal Cell Technology Unit, iBET, Instituto
            de Biologia Experimental e Tecnologica, Av. da Republica, Qta. do 
            Marques, ITQB/iBET building, Oeiras 2780-157, Portugal
REFERENCE   3  (bases 1 to 7482)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "G3 
            (Bethesda) (2021) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (11-NOV-2020) Animal Cell Technology Unit, iBET, Instituto de 
            Biologia Experimental e Tecnologica, Av. da Republica, Qta. do 
            Marques, ITQB/iBET building, Oeiras 2780-157, Portugal"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7482
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..48
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        207..225
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             253..969
                     /label=EGFP
                     /note="enhanced GFP"
     CDS             1075..1116
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
     CDS             1126..1143
                     /label=6xHis
                     /note="6xHis affinity tag"
     polyA_signal    1161..1290
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     primer_bind     1432..1448
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        1469..1951
                     /label=hr5 enhancer
                     /note="baculovirus early transcription enhancer"
     promoter        1955..2546
                     /label=IE1 promoter
                     /note="promoter of the ie1 gene from the baculovirus
                     Autographa californica"
     sig_peptide     2552..2611
                     /label=IgM signal sequence
                     /note="signal sequence from mouse IgM heavy chain"
     CDS             2624..2647
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
     CDS             2654..2668
                     /label=enterokinase site
                     /note="enterokinase recognition and cleavage site"
     CDS             2686..3981
                     /label=FLPo
                     /note="nuclear-targeted site-specific recombinase"
     CDS             4037..4063
                     /label=9xHis
                     /note="9xHis affinity tag"
     terminator      4085..4392
                     /label=IE1 terminator
                     /note="terminator of the ie1 gene from the baculovirus 
                     Autographa californica"
     regulatory      complement(4505..4553)
                     /regulatory_class="polyA_signal_sequence"
     CDS             complement(4620..5411)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     misc_recomb     5419..5466
     promoter        5515..5619
                     /label=AmpR promoter
     misc_feature    5804..6183
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene;
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     rep_origin      6652..7240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcgg atcctacctg
       61 gttctttccg cctcagaagc catacttaag ggctggctta actatgcggc atcagagcag
      121 attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac
      181 ataaccttat gtatcataca catacgattt aggtgacact atagaactcg agtgagcatc
      241 gatcccacga ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      301 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      361 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      421 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      481 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      541 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      601 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      661 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
      721 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
      781 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
      841 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
      901 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
      961 ctgtacaagt aaagcggccg caaagctagc ttcgatccag acatgataag atacattgat
     1021 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gcccgcggtt cgaaggtaag
     1081 cctatcccta accctctcct cggtctcgat tctacgcgta ccggtcatca tcaccatcac
     1141 cattgagttt atctgactaa atcttagttt gtattgtcat gttttaatac aatatgttat
     1201 gtttaaatat gtttttaata aattttataa aataatttca acttttattg taacaacatt
     1261 gtccatttac acactccttt caagcgcgtg ggatcggcga gtcagtgagc gaggaagcgg
     1321 agagcgccca atacgcaagg aaacagctat gaccatgtta atgcagctgg cacgacaggt
     1381 ttcccgactg gaaagcgggc agtgagcgga aggcccatga acaatttcac acaggaaaca
     1441 gctatgacca tgattacgaa ttcccgggcg cgtaaaacac aatcaagtat gagtcataag
     1501 ctgatgtcat gttttgcaca cggctcataa ccgaactggc tttacgagta gaattctact
     1561 tgtaacgcac gatcagtgga tgatgtcatt tgtttttcaa atcgagatga tgtcatgttt
     1621 tgcacacggc tcataaactc gctttacgag tagaattcta cgtgtaacgc acgatcgatt
     1681 gatgagtcat ttgttttgca atatgatatc atacaatatg actcatttgt ttttcaaaac
     1741 cgaacttgat ttacgggtag aattctactt gtaaagcaca atcaaaaaga tgatgtcatt
     1801 tgtttttcaa aactgaactc gctttacgag tagaattcta cgtgtaaaac acaatcaaga
     1861 aatgatgtca tttgttataa aaataaaagc tgatgtcatg ttttgcacat ggctcataac
     1921 taaactcgct ttacgggtag aattctacgc gcgtcgatgt ctttgtgatg cgcgcgacat
     1981 ttttgtaggt tattgataaa atgaacggat acgttgcccg acattatcat taaatccttg
     2041 gcgtagaatt tgtcgggtcc attgtccgtg tgcgctagca tgcccgtaac ggacctcgta
     2101 cttttggctt caaaggtttt gcgcacagac aaaatgtgcc acacttgcag ctctgcatgt
     2161 gtgcgcgtta ccacaaatcc caacggcgca gtgtacttgt tgtatgcaaa taaatctcga
     2221 taaaggcgcg gcgcgcgaat gcagctgatc acgtacgtcc ctcgtgttcc gttcaaggac
     2281 ggtgttatcg acctcagatt aatgtttatc ggccgactgt tttcgtatcc gctcaccaaa
     2341 cgcgtttttg cattaacatt gtatgtcggc ggatgttcta tatctaattt gaataaataa
     2401 acgataaccg cgttggtttt agagggcata ataaaagaaa tattgttatc gtgttcgcca
     2461 ttagggcagt ataaattgac gttcatgttg gatattgttt cagttgcaag ttgacactgg
     2521 cggcgacaag atcgtgaaca accaagtgac catgaaattc agctgggtca tgttcttcct
     2581 gatggcagtg gttacagggg tcaattcaga ggttcaggca agctggagcc acccgcagtt
     2641 cgaaaagggt gcagatgacg acgacaaggt accggatccg ctcccatggc tcccaagaaa
     2701 aagcgcaagg tcatgcccca gttcggtatc ctgtgcaaga ccccccccaa ggtgctcgtg
     2761 cgtcagttcg tcgagcgttt cgagcgcccc tccggcgaga agatcgcttc ctgcgctgct
     2821 gagctgacct acctgtgctg gatgatcacc cacaacggca ccgctatcaa gcgtgctacc
     2881 ttcatgtcct acaacaccat catctccaac tccctgtcct tcgacatcgt gaacaagtcc
     2941 ctgcagttca agtacaagac ccagaaggct accatcctcg aggcttccct gaagaagctg
     3001 atccccgctt gggagttcac catcatccct tactacggcc agaagcacca gtccgacatc
     3061 accgacatcg tgtcccgtct gcaactccag ttcgagtcct ccgaggaagc tgacaagggc
     3121 aactcccgtt ccaagaagat gctgaaggct ctgctgtccg agggcgagtc catctgggag
     3181 atcaccgaga agatcctgaa ctccttcgag tacacctccc gtttcaccaa gaccaagacc
     3241 ctgtaccagt tcctgttcct ggccaccttc atcaactgcg gtcgtttctc cgacatcaag
     3301 aacgtggacc ccaagtcctt caagctggtg cagaacaagt acctgggtgt catcatccag
     3361 tgcctggtca ccgagactaa gacctccgtg tcccgccaca tctacttctt ctccgctcgt
     3421 ggtcgtatcg accccctggt gtacctggac gagttcctgc gcaactccga gcccgtgctg
     3481 aagcgtgtga accgtaccgg caactcctcc agcaacaagc aagagtacca gctgctgaag
     3541 gacaacctcg tgcgctccta caacaaggct ctgaagaaga acgctcccta ctccatcttc
     3601 gctatcaaga acggtcccaa gtcccacatc ggtcgtcacc tgatgacatc gttcttgtcc
     3661 atgaagggcc tgaccgagct gaccaacgtc gtgggcaact ggtccgacaa gcgcgcttcc
     3721 gctgtggctc gtaccaccta cacccaccag atcaccgcta tccccgacca ctacttcgct
     3781 ctggtgtccc gttactacgc ttacgacccc atctccaagg aaatgatcgc tctgaaggac
     3841 gagactaacc ccatcgagga atggcagcac atcgagcagc tgaagggttc cgctgagggt
     3901 tccatccgtt accccgcttg gaacggtatc atctcccaag aggtgctgga ctacctgtcc
     3961 tcctacatca accgtcgtat cgtcgactag aaagcttgcg gccgcaggag ctccgggctt
     4021 ctcctcaatt tccgctcatc accaccatca tcaccatcac caccactaag tgattaacct
     4081 caggttatac atatattttg aatttaatta attatacata tattttatat tatttttgtc
     4141 ttttattatc gaggggccgt tgttggtgtg gggttttgca tagaaataac aatgggagtt
     4201 ggcgacgttg ctgcgccaac accacctccc ttccctcctt tcatcatgta tctgtagata
     4261 aaataaaata ttaaacctaa aaacaagacc gcgcctatca acaaaatgat aggcattaac
     4321 ttgccgctga cgctgtcact aacgttggac gatttgccga ctaaaccttc atcgcccagt
     4381 aaccaatcta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4441 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4501 attaaacaca aaaaaccaac acacagatgt aatgaaaata aagatatttt attgcggcca
     4561 tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg aaccccagag tcccgctcag
     4621 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg
     4681 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta
     4741 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca
     4801 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg
     4861 agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc
     4921 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt
     4981 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta
     5041 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat
     5101 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg
     5161 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct
     5221 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg
     5281 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc
     5341 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct
     5401 tgttcaatca tccatggaga agttcctatt ccgaagttcc tattcttcaa aaggtatagg
     5461 aacttcacta gtcccgatcc gtcgacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5521 acccctattt gtttattttt ttaaatacat tcaaatatgt atccgctcat gagacaataa
     5581 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5641 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5701 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5761 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5821 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5881 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5941 gaaaagcatc tcacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6001 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6061 gcttttttgc acaacatggg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6121 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6181 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6241 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6301 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6361 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6421 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6481 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6541 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6601 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6661 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6721 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6781 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6841 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6901 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6961 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7021 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7081 ggacaggtat ccggtcagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7141 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7201 atttttgtgg tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7261 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7321 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7381 agcgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     7441 gcctctcccc gcgcgttggc cgactcatta atgcagggcc gc
//
