LOCUS       sequence-12220-e        6665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6665)
  AUTHORS   Berrow NS, Alderton D, Sainsbury S, Nettleship J, Assenberg R, 
            Rahman N, Stuart DI, Owens RJ
  TITLE     A versatile ligation-independent cloning method suitable for 
            high-throughput expression screening applications.
  JOURNAL   Nucleic Acids Res. 2007  . 35(6):e45.
  PUBMED    17317681
REFERENCE   2  (bases 1 to 6665)
  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: "Nucleic 
            Acids Res. 2007  . 35(6):e45."
COMMENT     Sequence Label: pOPINM
FEATURES             Location/Qualifiers
     source          1..6665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1449..1726
                     /label=chicken beta-actin promoter
     primer_bind     2005..2027
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2089..2108
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2150..2169
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2150..2168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2169..2193
                     /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        2209..2318
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             2339..2356
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2366..3466
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     3440..3463
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    3540..3561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3576..3581,3582..3599,3600..3606)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3614..3630
                     /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)."
     primer_bind     3619..3641
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3638..3654
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3638..3654
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3650..3823
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(3670..3687)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3670..3686)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3679..3701)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3853..3870
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3960..3979)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4025..4080
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4079..4098)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4168..4215
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4228..4933
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4316..4688
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4345..4933)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4942..4975)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(5143..5730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5222..5241)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5743..6534,6535..6603))
                     /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     6366..6385
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg
     1021 taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct
     1081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1141 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1201 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1261 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1321 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1381 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1441 atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1501 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1561 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
     1621 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1681 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1741 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1801 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctccttcgg gctgtaatta
     1861 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1921 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc
     1981 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2041 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2101 ttattgtgct gtctcatcat tttggcaaag aattggatcg gaccgaaatt aatacgactc
     2161 actatagggg aattgtgagc ggataacaat tccccggagt taatccggga cctttaattc
     2221 aacccaacac aatatattat agttaaataa gaattattat caaatcattt gtatattaat
     2281 taaaatacta tactgtaaat tacattttat ttacaatcaa aggagatata ccatggcaca
     2341 ccatcaccac catcacagca gcggcatgaa aatcgaagaa ggtaaactgg taatctggat
     2401 taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac
     2461 cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc
     2521 ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc
     2581 tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc
     2641 gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga
     2701 agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga
     2761 gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct
     2821 gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta
     2881 tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg
     2941 tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc
     3001 catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc
     3061 atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa
     3121 gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc
     3181 gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga
     3241 agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt
     3301 ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc cagaaaggtg aaatcatgcc
     3361 gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc
     3421 cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactagca gcggtctgga
     3481 agttctgttt cagggtaccg tcgacccgac tggaaagcgg gcagtgagcg caacgcaatt
     3541 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     3601 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     3661 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     3721 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     3781 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc aagctttcta
     3841 gaccagttta aacatcacca tcaccatcac taagtgatta acctcaggtg caggctgcct
     3901 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcgat
     3961 ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     4021 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     4081 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     4141 agtttggcaa catatgccca tatgtaacta gcataacccc ttggggcctc taaacgggtc
     4201 ttgaggggtt ttttgctgaa agcatgcgga ggaaattctc cttgaagttt ccctggtgtt
     4261 caaagtaaag gagtttgcac cagacgcacc tctgttcact ggtccggcgt attaaaacac
     4321 gatacattgt tattagtaca tttattaagc gctagattct gtgcgttgtt gatttacaga
     4381 caattgttgt acgtatttta ataattcatt aaatttataa tctttagggt ggtatgttag
     4441 agcgaaaatc aaatgatttt cagcgtcttt atatctgaat ttaaatatta aatcctcaat
     4501 agatttgtaa aataggtttc gattagtttc aaacaagggt tgtttttccg aaccgatggc
     4561 tggactatct aatggatttt cgctcaacgc cacaaaactt gccaaatctt gtagcagcaa
     4621 tctagctttg tcgatattcg tttgtgtttt gttttgtaat aaaggttcga cgtcgttcaa
     4681 aatattatgc gcttttgtat ttctttcatc actgtcgtta gtgtacaatt gactcgacgt
     4741 aaacacgtta aatagagctt ggacatattt aacatcgggc gtgttagctt tattaggccg
     4801 attatcgtcg tcgtcccaac cctcgtcgtt agaagttgct tccgaagacg attttgccat
     4861 agccacacga cgcctattaa ttgtgtcggc taacacgtcc gcgatcaaat ttgtagttga
     4921 gctttttgga attgcgatcg cataacttcg tatagcatac attatacgaa gttataagct
     4981 cggaacgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5041 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5101 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5161 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5221 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5281 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc
     5341 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5401 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5461 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5521 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5581 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5641 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5701 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgttaccaat gcttaatcag
     5761 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5821 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5881 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5941 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6001 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6061 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6121 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6181 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6241 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6301 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6361 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6421 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6481 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6541 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6601 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgtccgc
     6661 gcgtt
//
