LOCUS       62056_12460        5100 bp DNA     circular SYN 24-MAY-2021
DEFINITION  Cloning vector phr5-AcIE1-iFlp, complete sequence.
ACCESSION   MW246025
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5100)
  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 5100)
  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 5100)
  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..5100
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..483
                     /label=hr5 enhancer
                     /note="baculovirus early transcription enhancer"
     promoter        487..1078
                     /label=IE1 promoter
                     /note="promoter of the ie1 gene from the baculovirus
                     Autographa californica"
     sig_peptide     1084..1143
                     /label=IgM signal sequence
                     /note="signal sequence from mouse IgM heavy chain"
     CDS             1156..1179
                     /codon_start=1
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
                     /translation="WSHPQFEK"
     CDS             1186..1200
                     /codon_start=1
                     /label=enterokinase site
                     /note="enterokinase recognition and cleavage site"
                     /translation="DDDDK"
     CDS             1218..2513
                     /codon_start=1
                     /label=FLPo
                     /note="nuclear-targeted site-specific recombinase"
                     /translation="MAPKKKRKVMPQFGILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYYGQKHQSDITDIVSRLQLQFESSEEADKGNSRSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     CDS             2569..2595
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     terminator      2617..2924
                     /label=IE1 terminator
                     /note="terminator of the ie1 gene from the baculovirus 
                     Autographa californica"
     promoter        2954..3058
                     /label=AmpR promoter
     CDS             3059..3916
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4090..4678
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    4966..4987
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5002..5032
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5040..5056
                     /label=lac operator
                     /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     5064..5080
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cgcgtaaaac acaatcaagt atgagtcata agctgatgtc atgttttgca cacggctcat
       61 aaccgaactg gctttacgag tagaattcta cttgtaacgc acgatcagtg gatgatgtca
      121 tttgtttttc aaatcgagat gatgtcatgt tttgcacacg gctcataaac tcgctttacg
      181 agtagaattc tacgtgtaac gcacgatcga ttgatgagtc atttgttttg caatatgata
      241 tcatacaata tgactcattt gtttttcaaa accgaacttg atttacgggt agaattctac
      301 ttgtaaagca caatcaaaaa gatgatgtca tttgtttttc aaaactgaac tcgctttacg
      361 agtagaattc tacgtgtaaa acacaatcaa gaaatgatgt catttgttat aaaaataaaa
      421 gctgatgtca tgttttgcac atggctcata actaaactcg ctttacgggt agaattctac
      481 gcgcgtcgat gtctttgtga tgcgcgcgac atttttgtag gttattgata aaatgaacgg
      541 atacgttgcc cgacattatc attaaatcct tggcgtagaa tttgtcgggt ccattgtccg
      601 tgtgcgctag catgcccgta acggacctcg tacttttggc ttcaaaggtt ttgcgcacag
      661 acaaaatgtg ccacacttgc agctctgcat gtgtgcgcgt taccacaaat cccaacggcg
      721 cagtgtactt gttgtatgca aataaatctc gataaaggcg cggcgcgcga atgcagctga
      781 tcacgtacgt ccctcgtgtt ccgttcaagg acggtgttat cgacctcaga ttaatgttta
      841 tcggccgact gttttcgtat ccgctcacca aacgcgtttt tgcattaaca ttgtatgtcg
      901 gcggatgttc tatatctaat ttgaataaat aaacgataac cgcgttggtt ttagagggca
      961 taataaaaga aatattgtta tcgtgttcgc cattagggca gtataaattg acgttcatgt
     1021 tggatattgt ttcagttgca agttgacact ggcggcgaca agatcgtgaa caaccaagtg
     1081 accatgaaat tcagctgggt catgttcttc ctgatggcag tggttacagg ggtcaattca
     1141 gaggttcagg caagctggag ccacccgcag ttcgaaaagg gtgcagatga cgacgacaag
     1201 gtaccggatc cgctcccatg gctcccaaga aaaagcgcaa ggtcatgccc cagttcggta
     1261 tcctgtgcaa gacccccccc aaggtgctcg tgcgtcagtt cgtcgagcgt ttcgagcgcc
     1321 cctccggcga gaagatcgct tcctgcgctg ctgagctgac ctacctgtgc tggatgatca
     1381 cccacaacgg caccgctatc aagcgtgcta ccttcatgtc ctacaacacc atcatctcca
     1441 actccctgtc cttcgacatc gtgaacaagt ccctgcagtt caagtacaag acccagaagg
     1501 ctaccatcct cgaggcttcc ctgaagaagc tgatccccgc ttgggagttc accatcatcc
     1561 cttactacgg ccagaagcac cagtccgaca tcaccgacat cgtgtcccgt ctgcaactcc
     1621 agttcgagtc ctccgaggaa gctgacaagg gcaactcccg ttccaagaag atgctgaagg
     1681 ctctgctgtc cgagggcgag tccatctggg agatcaccga gaagatcctg aactccttcg
     1741 agtacacctc ccgtttcacc aagaccaaga ccctgtacca gttcctgttc ctggccacct
     1801 tcatcaactg cggtcgtttc tccgacatca agaacgtgga ccccaagtcc ttcaagctgg
     1861 tgcagaacaa gtacctgggt gtcatcatcc agtgcctggt caccgagact aagacctccg
     1921 tgtcccgcca catctacttc ttctccgctc gtggtcgtat cgaccccctg gtgtacctgg
     1981 acgagttcct gcgcaactcc gagcccgtgc tgaagcgtgt gaaccgtacc ggcaactcct
     2041 ccagcaacaa gcaagagtac cagctgctga aggacaacct cgtgcgctcc tacaacaagg
     2101 ctctgaagaa gaacgctccc tactccatct tcgctatcaa gaacggtccc aagtcccaca
     2161 tcggtcgtca cctgatgaca tcgttcttgt ccatgaaggg cctgaccgag ctgaccaacg
     2221 tcgtgggcaa ctggtccgac aagcgcgctt ccgctgtggc tcgtaccacc tacacccacc
     2281 agatcaccgc tatccccgac cactacttcg ctctggtgtc ccgttactac gcttacgacc
     2341 ccatctccaa ggaaatgatc gctctgaagg acgagactaa ccccatcgag gaatggcagc
     2401 acatcgagca gctgaagggt tccgctgagg gttccatccg ttaccccgct tggaacggta
     2461 tcatctccca agaggtgctg gactacctgt cctcctacat caaccgtcgt atcgtcgact
     2521 agaaagcttg cggccgcagg agctccgggc ttctcctcaa tttccgctca tcaccaccat
     2581 catcaccatc accaccacta agtgattaac ctcaggttat acatatattt tgaatttaat
     2641 taattataca tatattttat attatttttg tcttttatta tcgaggggcc gttgttggtg
     2701 tggggttttg catagaaata acaatgggag ttggcgacgt tgctgcgcca acaccacctc
     2761 ccttccctcc tttcatcatg tatctgtaga taaaataaaa tattaaacct aaaaacaaga
     2821 ccgcgcctat caacaaaatg ataggcatta acttgccgct gacgctgtca ctaacgttgg
     2881 acgatttgcc gactaaacct tcatcgccca gtaaccaatc tagacgtcag gtggcacttt
     2941 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3001 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3061 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3121 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3181 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3241 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3301 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3361 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3421 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3481 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3541 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3601 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3661 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3721 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3781 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3841 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3901 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3961 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4021 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4081 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4141 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4201 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4261 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4321 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4381 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4441 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4501 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4561 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4621 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4681 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4741 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4801 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4861 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     4921 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     4981 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     5041 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcccggg
//
