LOCUS       sequence-54672-e        7013 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 7013)
  AUTHORS   Bird LE
  TITLE     High throughput construction and small scale expression screening of
            multi-tag vectors in Escherichia coli.
  JOURNAL   Methods. 2011 Sep;55(1):29-37. Epub 2011 Aug 10.
  PUBMED    21856427
REFERENCE   2  (bases 1 to 7013)
  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: "Methods."; 
            date: "2011-09"; volume: "55(1)"; pages: "29-37. Epub 2011 Aug 10"
COMMENT     Sequence Label: pOPINNUSA
FEATURES             Location/Qualifiers
     source          1..7013
                     /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             2357..3838
                     /codon_start=1
                     /gene="nusA"
                     /product="transcription elongation protein (N utilization 
                     substance protein A)"
                     /label=NusA
                     /note="highly soluble in E. coli"
                     /translation="NKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVR
                     VQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRI
                     TTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILR
                     EDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEI
                     KAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQTVSTELGGERIDIVLWDDNPAQ
                     FVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTV
                     DDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLDE
                     PTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDL
                     AEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     protein_bind    3912..3933
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3948..3953,3954..3971,3972..3978)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3986..4002
                     /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     3991..4013
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4010..4026
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4010..4026
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4022..4195
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(4042..4059)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4042..4058)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4051..4073)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4308..4327)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4373..4428
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4427..4446)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4516..4563
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4576..5281
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4664..5036
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4693..5281)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5290..5323)
                     /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(5491..6078)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5570..5589)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6091..6882,6883..6951))
                     /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     6714..6733
                     /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 catcacaaca aagaaatttt ggctgtagtt gaagccgtat ccaatgaaaa
     2401 ggcgctacct cgcgagaaga ttttcgaagc attggaaagc gcgctggcga cagcaacaaa
     2461 gaaaaaatat gaacaagaga tcgacgtccg cgtacagatc gatcgcaaaa gcggtgattt
     2521 tgacaccttc cgtcgctggt tagttgttga tgaagtcacc cagccgacca aggaaatcac
     2581 ccttgaagcc gcacgttatg aagatgaaag cctgaacctg ggcgattacg ttgaagatca
     2641 gattgagtct gttacctttg accgtatcac tacccagacg gcaaaacagg ttatcgtgca
     2701 gaaagtgcgt gaagccgaac gtgcgatggt ggttgatcag ttccgtgaac acgaaggtga
     2761 aatcatcacc ggcgtggtga aaaaagtaaa ccgcgacaac atctctctgg atctgggcaa
     2821 caacgctgaa gccgtgatcc tgcgtgaaga tatgctgccg cgtgaaaact tccgccctgg
     2881 cgaccgcgtt cgtggcgtgc tttattccgt tcgcccggaa gcgcgtggcg cgcaactgtt
     2941 cgtcactcgt tccaagccgg aaatgctgat cgaactgttc cgtattgaag tgccagaaat
     3001 cggcgaagaa gtgattgaaa ttaaagcagc ggctcgcgat ccgggttctc gtgcgaaaat
     3061 cgcggtgaaa accaacgata aacgtatcga tccggtaggt gcttgcgtag gtatgcgtgg
     3121 cgcgcgtgtt cagacggtgt ctactgaact gggtggcgag cgtatcgata tcgtcctgtg
     3181 ggatgataac ccggcgcagt tcgtgattaa cgcaatggca ccggcagacg ttgcttctat
     3241 cgtggtggat gaagataaac acaccatgga tatcgccgtt gaagccggta acctggcgca
     3301 ggcgattggc cgtaacggtc agaacgtgcg tctggcttcg cagctgagcg gttgggaact
     3361 caacgtgatg accgttgacg acctgcaggc taagcatcag gcggaagcgc acgcagcgat
     3421 cgacaccttc accaaatatc tcgacatcga cgaagacttc gcgactgttc tggtagaaga
     3481 aggcttctcg acgctggaag aactggccta tgtgccgatg aaagagctgt tggaaatcga
     3541 aggccttgat gagccgaccg ttgaagcact gcgcgagcgt gctaaaaatg cactggccac
     3601 cattgcacag gcccaggaag aaagcctcgg tgataacaaa ccggctgacg atctgctgaa
     3661 ccttgaaggg gtagatcgtg atttggcatt caaactggcc gcccgtggcg tttgtacgct
     3721 ggaagatctc gccgaacagg gcattgatga tctggctgat atcgaagggt tgaccgacga
     3781 aaaagccgga gcactgatta tggctgcccg taatatttgc tggttcggtg acgaagcgag
     3841 cagcggtctg gaagttctgt ttcagggtac cgtcgacccg actggaaagc gggcagtgag
     3901 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     3961 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     4021 tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     4081 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     4141 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     4201 ccaagctttc tagaccagtt tgtgattaac ctcaggtgca ggctgcctat cagaaggtgg
     4261 tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcgatct ttttccctct
     4321 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     4381 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     4441 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     4501 tatgcccata tgtaactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     4561 ttgctgaaag catgcggagg aaattctcct tgaagtttcc ctggtgttca aagtaaagga
     4621 gtttgcacca gacgcacctc tgttcactgg tccggcgtat taaaacacga tacattgtta
     4681 ttagtacatt tattaagcgc tagattctgt gcgttgttga tttacagaca attgttgtac
     4741 gtattttaat aattcattaa atttataatc tttagggtgg tatgttagag cgaaaatcaa
     4801 atgattttca gcgtctttat atctgaattt aaatattaaa tcctcaatag atttgtaaaa
     4861 taggtttcga ttagtttcaa acaagggttg tttttccgaa ccgatggctg gactatctaa
     4921 tggattttcg ctcaacgcca caaaacttgc caaatcttgt agcagcaatc tagctttgtc
     4981 gatattcgtt tgtgttttgt tttgtaataa aggttcgacg tcgttcaaaa tattatgcgc
     5041 ttttgtattt ctttcatcac tgtcgttagt gtacaattga ctcgacgtaa acacgttaaa
     5101 tagagcttgg acatatttaa catcgggcgt gttagcttta ttaggccgat tatcgtcgtc
     5161 gtcccaaccc tcgtcgttag aagttgcttc cgaagacgat tttgccatag ccacacgacg
     5221 cctattaatt gtgtcggcta acacgtccgc gatcaaattt gtagttgagc tttttggaat
     5281 tgcgatcgca taacttcgta tagcatacat tatacgaagt tataagctcg gaacgctgcg
     5341 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5401 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5461 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc tgacgagcat
     5521 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5581 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5641 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     5701 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5761 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5821 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5881 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5941 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6001 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6061 agaaaaaaag gatctcaaga agatcctttg ttaccaatgc ttaatcagtg aggcacctat
     6121 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6181 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6241 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6301 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6361 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6421 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6481 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6541 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6601 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6661 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6721 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6781 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6841 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6901 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6961 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgtccgcgc gtt
//
