LOCUS       sequence-481077-        6298 bp    DNA     circular SYN 29-JUL-2025
DEFINITION  Expresses LPETG-tagged Cre recombinase in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6298)
  AUTHORS   Eweje F, Ibrahim V, Shajii A, Walsh ML, Ahmad K, Alrefai A, Miyasato
            D, Davis JR, Ham H, Li K, Roehrl M, Haller CA, Liu DR, Chen J, 
            Chaikof EL
  TITLE     Self-assembling protein nanoparticles for cytosolic delivery of 
            nucleic acids and proteins.
  JOURNAL   Nat Biotechnol. 2025 May 15. doi: 10.1038/s41587-025-02664-2.
  PUBMED    40374955
REFERENCE   2  (bases 1 to 6298)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 29, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 May 15. doi: 10.1038/s41587-025-02664-2."
COMMENT     Sequence Label: pET-Cre-LPETG-6xHis
FEATURES             Location/Qualifiers
     source          1..6298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             71..1093
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     G"
     CDS             1211..1228
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1247..1291
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1329..1347)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1343..1390
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1427..1882
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1514..1533)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1724..1745
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1997..2788,2789..2857))
                     /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     2620..2639
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2858..2950)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3031..3619
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3520..3539
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3773..3790
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3805..3944
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3870..3889)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4030..4052
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4046..4237)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4809..5891)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5853..5872
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5892..5969)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6175..6194
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6282..6298,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6282..6298,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgaatttac tgaccgtaca ccaaaatttg cctgcattgc cggtcgatgc
      121 aacgagtgat gaggttcgca agaacctgat ggacatgttc agggatcgcc aggcgttttc
      181 tgagcatacc tggaaaatgc ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt
      241 gaataaccgg aaatggtttc ccgcagaacc tgaagatgtt cgcgattatc ttctatatct
      301 tcaggcgcgc ggtctggcag taaaaactat ccagcaacat ttgggccagc taaacatgct
      361 tcatcgtcgg tccgggctgc cacgaccaag tgacagcaat gctgtttcac tggttatgcg
      421 gcgtatccga aaagaaaacg ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga
      481 acgcactgat ttcgaccagg ttcgttcact catggaaaat agcgatcgct gccaggatat
      541 acgtaatctg gcatttctgg ggattgctta taacaccctg ttacgtatag ccgaaattgc
      601 caggatcagg gttaaagata tctcacgtac tgacggtggg agaatgttaa tccatattgg
      661 cagaacgaaa acgctggtta gcaccgcagg tgtagagaag gcacttagcc tgggggtaac
      721 taaactggtc gagcgatgga tttccgtctc tggtgtagct gatgatccga ataactacct
      781 gttttgccgg gtcagaaaaa atggtgttgc cgcgccatct gccaccagcc agctatcaac
      841 tcgcgccctg gaagggattt ttgaagcaac tcatcgattg atttacggcg ctaaggatga
      901 ctctggtcag agatacctgg cctggtctgg acacagtgcc cgtgtcggag ccgcgcgaga
      961 tatggcccgc gctggagttt caataccgga gatcatgcaa gctggtggct ggaccaatgt
     1021 aaatattgtc atgaactata tccgtaacct ggatagtgaa acaggggcaa tggtgcgcct
     1081 gctggaagat ggcaccggtg gtagcggtgg ttctggtggt tctggtggta gcggcggtag
     1141 cggtggtagc ggtggtagcg gtggcagcgg cggtaccctc ccagagaccg gtgtcgtaga
     1201 cggcggatcc catcaccacc accatcacta atgactcgag tctggtaaag aaaccgctgc
     1261 tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc
     1321 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1381 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1441 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1501 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1561 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1621 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1681 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1741 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1801 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1861 aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct
     1921 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     1981 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2041 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2101 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     2161 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     2221 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     2281 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     2341 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     2401 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2461 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     2521 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     2581 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     2641 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     2701 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     2761 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     2821 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat
     2881 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2941 aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3001 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3061 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3121 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3181 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3241 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3301 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3361 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3421 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3481 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3541 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3601 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3661 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3721 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3781 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3841 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3901 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3961 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4021 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4081 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4141 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4201 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4261 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4321 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4381 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4441 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4501 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4561 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4621 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4681 ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4741 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4801 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4861 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4921 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4981 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     5041 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     5101 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     5161 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5221 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     5281 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     5341 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5401 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5461 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5521 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5581 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5641 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5701 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5761 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5821 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5881 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5941 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     6001 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     6061 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     6121 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     6181 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     6241 tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactata
//
