LOCUS       Exported                6783 bp ds-DNA     circular SYN 18-JAN-2022
DEFINITION  Expresses Cre recombinase protein in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    CRE recombinase
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6783)
  AUTHORS   D'Astolfo DS, Pagliero RJ, Pras A, Karthaus WR, Clevers H, Prasad V,
            Lebbink RJ, Rehmann H, Geijsen N
  TITLE     Efficient Intracellular Delivery of Native Proteins.
  JOURNAL   Cell. 2015 Apr 23;161(3):674-690. doi: 10.1016/j.cell.2015.03.028.
  PUBMED    25910214
REFERENCE   2  (bases 1 to 6783)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 18, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6783
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(45..63)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        131..235
                     /gene="bla"
                     /label=AmpR promoter
     CDS             236..1096
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(454..473)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1267..1855
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1756..1775
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2009..2026
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2041..2180
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2106..2125)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2266..2288
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2282..2473)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3785..4867)
                     /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     4829..4848
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4868..4945)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5151..5170
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5254..5273
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5254..5272
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5273..5297
                     /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             5312..5334
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5353..5370
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5380..5397
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5437..5457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6537..6555)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6551..6598
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(6773..18)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct
       61 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
      121 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
      181 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
      241 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
      301 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
      361 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
      421 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
      481 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
      541 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
      601 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
      661 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
      721 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
      781 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
      841 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
      901 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
      961 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     1021 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     1081 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     1141 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     1201 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1261 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     1321 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     1381 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     1441 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     1501 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     1561 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     1621 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     1681 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     1741 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     1801 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     1861 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     1921 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     1981 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     2041 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     2101 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     2161 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2221 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2281 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     2341 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     2401 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     2461 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     2521 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     2581 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     2641 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     2701 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     2761 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     2821 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     2881 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     2941 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
     3001 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg
     3061 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     3121 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     3181 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
     3241 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
     3301 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
     3361 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
     3421 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
     3481 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     3541 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     3601 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     3661 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     3721 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     3781 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3841 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     3901 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     3961 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4021 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     4081 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     4141 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     4201 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     4261 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     4321 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     4381 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     4441 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     4501 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     4561 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     4621 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     4681 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     4741 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     4801 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     4861 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     4921 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     4981 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5041 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     5101 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     5161 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     5221 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     5281 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
     5341 atgggctcat cgcatcatca tcatcatcat tcatcgggtc tggttccgcg cggttcacac
     5401 ggtggtggtt cggcggccgc tatgggcacc cgcctgccga aaaaaaaacg caaagtgtcc
     5461 aacctgctga cggtgcacca gaatctgccg gcactgccgg tcgatgcaac cagtgacgaa
     5521 gtgcgcaaaa atctgatgga tatgtttcgt gaccgccaag ccttcagcga acatacgtgg
     5581 aaaatgctgc tgtcggtttg ccgtagctgg gcggcctggt gtaaactgaa caatcgcaaa
     5641 tggtttccgg cagaaccgga agatgtgcgt gactatctgc tgtacctgca ggcacgtggt
     5701 ctggcagtta aaaccatcca gcaacatctg ggccaactga acatgctgca ccgtcgctct
     5761 ggtctgccgc gtccgagtga ttccaatgcc gtcagtctgg tgatgcgtcg cattcgtaaa
     5821 gaaaacgtgg atgcaggcga acgcgctaaa caggcactgg cttttgaacg taccgatttc
     5881 gaccaagttc gctctctgat ggaaaacagt gatcgttgcc aggacatccg caatctggca
     5941 ttcctgggta ttgcttataa caccctgctg cgcattgcag aaatcgctcg tattcgcgtg
     6001 aaagatatca gccgtacgga cggcggtcgc atgctgattc acatcggccg taccaaaacg
     6061 ctggtttcca ccgcgggcgt cgaaaaagcc ctgtcactgg gtgtcacgaa actggtggaa
     6121 cgctggattt cagtttcggg cgtcgcagat gacccgaaca attacctgtt ttgtcgtgtg
     6181 cgcaaaaatg gtgttgcagc tccgagcgct acctctcagc tgagtacgcg tgcgctggaa
     6241 ggcatcttcg aagccaccca tcgcctgatt tatggcgcga aagatgacag cggtcagcgt
     6301 tacctggcat ggtccggtca ctcagctcgt gttggtgcag cacgtgatat ggcacgtgca
     6361 ggtgtctcta tcccggaaat tatgcaggcc ggcggttgga cgaacgtgaa tattgttatg
     6421 aactatattc gtaacctgga ctctgaaacg ggtgcgatgg tgcgtctgct ggaagatggc
     6481 gactgaggat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     6541 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     6601 gaaaggagga actatatccg gatatcccgc aagaggcccg gcagtaccgg cataaccaag
     6661 cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc attgttagat
     6721 ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc gcattaaagc
     6781 tta
//
