LOCUS       sequence-106328-        6199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Cre-6xHis in bacterial of cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6199)
  AUTHORS   Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder
            ML, Joung JK, Chen ZY, Liu DR
  TITLE     Cationic lipid-mediated delivery of proteins enables efficient 
            protein-based genome editing in vitro and in vivo.
  JOURNAL   Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 
            2014 Oct 30.
  PUBMED    25357182
REFERENCE   2  (bases 1 to 6199)
  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"; doi: "10.1038/nbt.3081"; 
            journalName: "Nat Biotechnol"; date: "2015-01"; volume: "33"; issue:
            "1"; pages: "73-80"
COMMENT     Sequence Label: pET-Cre-6xHis
FEATURES             Location/Qualifiers
     source          1..6199
                     /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..1099
                     /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="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             1109..1126
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1145..1189
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1227..1245)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1241..1288
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1325..1780
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1412..1431)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1622..1643
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1895..2686,2687..2755))
                     /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     2518..2537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2756..2848)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2929..3517
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3418..3437
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3671..3688
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3703..3845
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3771..3790)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3931..3953
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3947..4138)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4710..5792)
                     /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     5754..5773
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5793..5870)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6076..6095
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6183..6199,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6183..6199,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgagcaatt tactgaccgt acaccaaaat ttgcctgcat tgccggtcga
      121 tgcaacgagt gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt
      181 ttctgagcat acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa
      241 gttgaataac cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata
      301 tcttcaggcg cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat
      361 gcttcatcgt cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat
      421 gcggcgtatc cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt
      481 cgaacgcact gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga
      541 tatacgtaat ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat
      601 tgccaggatc agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat
      661 tggcagaacg aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt
      721 aactaaactg gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta
      781 cctgttttgc cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc
      841 aactcgcgcc ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga
      901 tgactctggt cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg
      961 agatatggcc cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa
     1021 tgtaaatatt gtcatgaact atatccgtaa cctggatagt gaaacagggg caatggtgcg
     1081 cctgctggaa gatggcgacg gcggatccca tcaccaccac catcactaat gactcgagtc
     1141 tggtaaagaa accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc
     1201 agcttaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1261 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1321 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1381 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1441 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1501 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1561 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1621 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1681 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1741 tttaacgcga attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga
     1801 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     1861 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     1921 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     1981 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     2041 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     2101 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     2161 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     2221 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     2281 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     2341 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     2401 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     2461 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     2521 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     2581 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     2641 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     2701 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     2761 tcctttttca atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata
     2821 tttgaatgta tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt
     2881 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2941 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3001 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3061 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3121 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3181 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3241 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3301 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3361 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3421 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3481 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3541 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3601 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3661 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3721 ccttacgcat ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc
     3781 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3841 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3901 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3961 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     4021 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     4081 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     4141 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     4201 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     4261 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     4321 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4381 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4441 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4501 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4561 ctagccgggt cctcaacgac aggagcacga tcatgctagt catgccccgc gcccaccgga
     4621 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4681 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4741 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4801 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     4861 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4921 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     4981 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5041 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5101 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5161 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5221 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5281 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5341 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5401 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5461 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5521 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5581 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5641 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     5701 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     5761 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     5821 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     5881 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     5941 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     6001 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     6061 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     6121 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa
     6181 attaatacga ctcactata
//
