LOCUS       Exported                6199 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression plasmid for wild-type Cre with an N-terminal 
            His-tag..
ACCESSION   .
VERSION     .
KEYWORDS    pET-His-Cre
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6199)
  AUTHORS   Jeffrey L. Bessen, Lena K. Afeyan, Vlado Dancik, Luke W. Koblan, 
  TITLE     High-resolution specificity profiling and off-target prediction for 
            site-specific DNA recombinases
  JOURNAL   Nature Communications volume 10, Article number: 1937 (2019)
REFERENCE   2  (bases 1 to 6199)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6199
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     97..116
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        97..115
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    116..140
                     /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             155..177
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             190..207
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             220..1245
                     /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="NLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLS
                     VCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPR
                     PSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLG
                     IAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERW
                     ISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRY
                     LAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDG
                     D"
     CDS             1261..1305
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1343..1361)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1357..1404
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1441..1896
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1528..1547)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1738..1759
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2011..2871)
                     /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     2634..2653
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2872..2964)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3045..3633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3534..3553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3787..3804
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3819..3958
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3884..3903)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4044..4066
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4060..4251)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4823..5905)
                     /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     5867..5886
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5906..5983)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6189..9
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
       61 cgtagaggat cgagatcgat ctcgatcccg cgaaattaat acgactcact ataggggaat
      121 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
      181 accatggcgc atcaccacca ccatcacggc gggagcagca atttactgac cgtacaccaa
      241 aatttgcctg cattgccggt cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac
      301 atgttcaggg atcgccaggc gttttctgag catacctgga aaatgcttct gtccgtttgc
      361 cggtcgtggg cggcatggtg caagttgaat aaccggaaat ggtttcccgc agaacctgaa
      421 gatgttcgcg attatcttct atatcttcag gcgcgcggtc tggcagtaaa aactatccag
      481 caacatttgg gccagctaaa catgcttcat cgtcggtccg ggctgccacg accaagtgac
      541 agcaatgctg tttcactggt tatgcggcgt atccgaaaag aaaacgttga tgccggtgaa
      601 cgtgcaaaac aggctctagc gttcgaacgc actgatttcg accaggttcg ttcactcatg
      661 gaaaatagcg atcgctgcca ggatatacgt aatctggcat ttctggggat tgcttataac
      721 accctgttac gtatagccga aattgccagg atcagggtta aagatatctc acgtactgac
      781 ggtgggagaa tgttaatcca tattggcaga acgaaaacgc tggttagcac cgcaggtgta
      841 gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc gatggatttc cgtctctggt
      901 gtagctgatg atccgaataa ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg
      961 ccatctgcca ccagccagct atcaactcgc gccctggaag ggatttttga agcaactcat
     1021 cgattgattt acggcgctaa ggatgactct ggtcagagat acctggcctg gtctggacac
     1081 agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg gagtttcaat accggagatc
     1141 atgcaagctg gtggctggac caatgtaaat attgtcatga actatatccg taacctggat
     1201 agtgaaacag gggcaatggt gcgcctgctg gaagatggcg actaatgact cgagtctggt
     1261 aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg actcgtctac tagcgcagct
     1321 taattaacct aggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1381 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1441 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1501 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1561 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1621 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1681 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1741 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1801 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1861 acgcgaattt taacaaaata ttaacgttta caatttctgg cggcacgatg gcatgagatt
     1921 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     1981 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2041 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2101 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     2161 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     2221 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     2281 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     2341 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2401 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     2461 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     2521 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     2581 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     2641 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     2701 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     2761 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     2821 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     2881 ttttcaatca tgattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     2941 aatgtattta gaaaaataaa caaataggtc atgaccaaaa tcccttaacg tgagttttcg
     3001 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3061 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3121 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3181 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3241 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3301 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3361 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3421 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3481 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3541 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3601 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3661 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3721 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3781 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3841 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     3901 ccgcatagtt aagccagtat acactcagct atcgctacgt gactgggtca tggctgcgcc
     3961 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4021 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4081 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4141 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4201 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4261 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4321 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4381 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4441 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4501 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4561 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4621 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4681 ggtcctcaac gacaggagca cgatcatgct agtcatgccc cgcgcccacc ggaaggagct
     4741 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4801 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4861 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4921 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4981 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     5041 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat ccaactaccg
     5101 agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5161 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5221 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5281 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     5341 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5401 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5461 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5521 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5581 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5641 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5701 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5761 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5821 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5881 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5941 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     6001 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     6061 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     6121 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     6181 tccacatcgg tgatgtcgg
//
