LOCUS       sequence-54667-e        6824 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 6824)
  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 6824)
  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: pOPINTF
FEATURES             Location/Qualifiers
     source          1..6824
                     /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..3649
                     /codon_start=1
                     /product="ribosome-associated chaperone from E. coli 
                     (Hoffmann et al., 2010)"
                     /label=Trigger Factor
                     /translation="QVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRK
                     GKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTY
                     SVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRV
                     TIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHA
                     ENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAI
                     RNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQA
                     KRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRN
                     VALEEQAVEAVLAKAKVTEKETTFNELMNQQA"
     protein_bind    3723..3744
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3759..3764,3765..3782,3783..3789)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3797..3813
                     /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     3802..3824
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3821..3837
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3821..3837
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3833..4006
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(3853..3870)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3853..3869)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3862..3884)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4119..4138)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4184..4239
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4238..4257)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4327..4374
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4387..5092
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4475..4847
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4504..5092)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5101..5134)
                     /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(5302..5889)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5381..5400)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5902..6693,6694..6762))
                     /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     6525..6544
                     /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 catcaccaag tttcagttga aaccactcaa ggccttggcc gccgtgtaac
     2401 gattactatc gctgctgaca gcatcgagac cgctgttaaa agcgagctgg tcaacgttgc
     2461 gaaaaaagta cgtattgacg gcttccgcaa gggcaaagtg ccaatgaata tcgttgctca
     2521 gcgttatggc gcgtctgtac gccaggacgt tctgggtgac ctgatgagcc gtaacttcat
     2581 tgacgccatc attaaagaaa aaatcaatcc ggctggcgca ccgacttatg ttccgggcga
     2641 atacaagctg ggtgaagact tcacttactc tgtagagttt gaagtttatc cggaagttga
     2701 actgcaaggt ctggaagcga tcgaagttga aaaaccgatc gttgaagtga ccgacgctga
     2761 cgttgacggc atgctggata ctctgcgtaa acagcaggcg acctggaaag aaaaagacgg
     2821 cgctgttgaa gcagaagacc gcgtgaccat cgacttcacc ggttctgtag acggcgaaga
     2881 gttcgaaggc ggtaaagcgt ctgatttcgt actggcgatg ggccagggtc gtatgatccc
     2941 gggctttgaa gacggtatca aaggccacaa agctggcgaa gagttcacca tcgacgtgac
     3001 cttcccggaa gaataccacg cagaaaacct gaaaggtaaa gcagcgaaat tcgctatcaa
     3061 cctgaagaaa gttgaagagc gtgaactgcc ggaactgacc gcagagttca tcaaacgttt
     3121 cggcgttgaa gatggttccg tagaaggtct gcgcgctgaa gtgcgtaaaa acatggagcg
     3181 cgagctgaag agcgccatcc gtaaccgcgt taagtctcag gcgatcgaag gtctggtaaa
     3241 agctaacgac atcgacgtac cggctgcgct gatcgacagc gaaatcgacg ttctgcgtcg
     3301 ccaggctgca cagcgtttcg gtggcaacga aaaacaagct ctggaactgc cgcgcgaact
     3361 gttcgaagaa caggctaaac gccgcgtagt tgttggcctg ctgctgggcg aagttatccg
     3421 caccaacgag ctgaaagctg acgaagagcg cgtgaaaggc ctgatcgaag agatggcttc
     3481 tgcgtacgaa gatccgaaag aagttatcga gttctacagc aaaaacaaag aactgatgga
     3541 caacatgcgc aatgttgctc tggaagaaca ggctgttgaa gctgtactgg cgaaagcgaa
     3601 agtgactgaa aaagaaacca ctttcaacga gctgatgaac cagcaggcga gcagcggtct
     3661 ggaagttctg tttcagggta ccgtcgaccc gactggaaag cgggcagtga gcgcaacgca
     3721 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     3781 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     3841 gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     3901 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     3961 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gccaagcttt
     4021 ctagaccagt ttgtgattaa cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
     4081 tggccaatgc cctggctcac aaataccact gagatcgatc tttttccctc tgccaaaaat
     4141 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     4201 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     4261 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat
     4321 atgtaactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     4381 gcatgcggag gaaattctcc ttgaagtttc cctggtgttc aaagtaaagg agtttgcacc
     4441 agacgcacct ctgttcactg gtccggcgta ttaaaacacg atacattgtt attagtacat
     4501 ttattaagcg ctagattctg tgcgttgttg atttacagac aattgttgta cgtattttaa
     4561 taattcatta aatttataat ctttagggtg gtatgttaga gcgaaaatca aatgattttc
     4621 agcgtcttta tatctgaatt taaatattaa atcctcaata gatttgtaaa ataggtttcg
     4681 attagtttca aacaagggtt gtttttccga accgatggct ggactatcta atggattttc
     4741 gctcaacgcc acaaaacttg ccaaatcttg tagcagcaat ctagctttgt cgatattcgt
     4801 ttgtgttttg ttttgtaata aaggttcgac gtcgttcaaa atattatgcg cttttgtatt
     4861 tctttcatca ctgtcgttag tgtacaattg actcgacgta aacacgttaa atagagcttg
     4921 gacatattta acatcgggcg tgttagcttt attaggccga ttatcgtcgt cgtcccaacc
     4981 ctcgtcgtta gaagttgctt ccgaagacga ttttgccata gccacacgac gcctattaat
     5041 tgtgtcggct aacacgtccg cgatcaaatt tgtagttgag ctttttggaa ttgcgatcgc
     5101 ataacttcgt atagcataca ttatacgaag ttataagctc ggaacgctgc gctcggtcgt
     5161 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5221 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5281 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc ctgacgagca tcacaaaaat
     5341 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5401 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5461 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     5521 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5581 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5641 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5701 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5761 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5821 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5881 ggatctcaag aagatccttt gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5941 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6001 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6061 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6121 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6181 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6241 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6301 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6361 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6421 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6481 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6541 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6601 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6661 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6721 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6781 ttattgaagc atttatcagg gttattgtct catgtccgcg cgtt
//
