LOCUS       sequence-12218-e        5531 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5531)
  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 5531)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pOPINF
FEATURES             Location/Qualifiers
     source          1..5531
                     /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"
     protein_bind    2430..2451
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2466..2471,2472..2489,2490..2496)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2504..2520
                     /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     2509..2531
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2528..2544
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2528..2544
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2540..2713
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(2560..2577)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2560..2576)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2569..2591)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2826..2845)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2891..2946
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2945..2964)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3034..3081
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3094..3799
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3182..3554
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3211..3799)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(3808..3841)
                     /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(4009..4596)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4088..4107)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4609..5400,5401..5469))
                     /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     5232..5251
                     /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 gcggtctgga agttctgttt cagggtaccg tcgacccgac
     2401 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     2461 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     2521 tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg
     2581 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2641 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2701 cctgaatggc gaatggcgcc aagctttcta gaccagtttg tgattaacct caggtgcagg
     2761 ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag
     2821 atcgatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     2881 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     2941 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     3001 gtttagagtt tggcaacata tgcccatatg taactagcat aaccccttgg ggcctctaaa
     3061 cgggtcttga ggggtttttt gctgaaagca tgcggaggaa attctccttg aagtttccct
     3121 ggtgttcaaa gtaaaggagt ttgcaccaga cgcacctctg ttcactggtc cggcgtatta
     3181 aaacacgata cattgttatt agtacattta ttaagcgcta gattctgtgc gttgttgatt
     3241 tacagacaat tgttgtacgt attttaataa ttcattaaat ttataatctt tagggtggta
     3301 tgttagagcg aaaatcaaat gattttcagc gtctttatat ctgaatttaa atattaaatc
     3361 ctcaatagat ttgtaaaata ggtttcgatt agtttcaaac aagggttgtt tttccgaacc
     3421 gatggctgga ctatctaatg gattttcgct caacgccaca aaacttgcca aatcttgtag
     3481 cagcaatcta gctttgtcga tattcgtttg tgttttgttt tgtaataaag gttcgacgtc
     3541 gttcaaaata ttatgcgctt ttgtatttct ttcatcactg tcgttagtgt acaattgact
     3601 cgacgtaaac acgttaaata gagcttggac atatttaaca tcgggcgtgt tagctttatt
     3661 aggccgatta tcgtcgtcgt cccaaccctc gtcgttagaa gttgcttccg aagacgattt
     3721 tgccatagcc acacgacgcc tattaattgt gtcggctaac acgtccgcga tcaaatttgt
     3781 agttgagctt tttggaattg cgatcgcata acttcgtata gcatacatta tacgaagtta
     3841 taagctcgga acgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3901 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3961 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4021 ccgccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4081 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4141 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4201 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4261 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4321 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4381 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4441 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4501 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4561 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgtt accaatgctt
     4621 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4681 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4741 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4801 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4861 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4921 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4981 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5041 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5101 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5161 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5221 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5281 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5341 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5401 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5461 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5521 gtccgcgcgt t
//
