LOCUS       Exported                5422 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pET GFP domain 11 LIC cloning vector (GFP C-11)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5422)
  TITLE     C-terminal fluorescent fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 5422)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5422
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     437..456
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     521..540
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      651..1239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1140..1159
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1393..1410
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1425..1567
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1493..1512)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1653..1675
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1669..1860)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2669..3751)
                     /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     3713..3732
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3752..3829)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4035..4054
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4138..4157
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4138..4156
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4157..4181
                     /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             4196..4218
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4231..4275
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4276..4293
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4294..4314
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4351..4398
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             4420..4437
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4490..4508)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4504..4551
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4588..5043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4675..4694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4885..4906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5136..529)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
       61 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
      121 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atgttttcac
      181 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
      241 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
      301 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
      361 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
      421 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
      481 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
      541 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
      601 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      661 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      721 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      781 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
      841 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      901 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      961 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1021 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1081 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1141 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1201 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1261 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1321 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     1381 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     1441 ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct
     1501 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     1561 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     1621 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     1681 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     1741 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     1801 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     1861 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     1921 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     1981 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     2041 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     2101 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     2161 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     2221 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     2281 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     2341 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     2401 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     2461 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     2521 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     2581 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     2641 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2701 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2761 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     2821 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     2881 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     2941 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     3001 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     3061 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     3121 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     3181 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     3241 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     3301 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     3361 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     3421 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     3481 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     3541 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     3601 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     3661 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     3721 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     3781 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     3841 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     3901 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     3961 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     4021 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     4081 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     4141 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     4201 ttaactttaa gaaggagata taccatggga ggcctgaacg atatttttga agcgcagaaa
     4261 attgaatggc atgaacacca tcaccatcac catgaaaacc tgtacttcca atccaatatt
     4321 ggtagtggga gcaacggcag cagcggatcc cgcgatcaca tggtcctgca cgagtacgtg
     4381 aacgccgccg ggatcactta gtaagcggcc gcactcgagc accaccacca ccaccactga
     4441 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     4501 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     4561 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     4621 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4681 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4741 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4801 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4861 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     4921 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4981 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaactagta acgtttacaa
     5041 tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5101 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     5161 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     5221 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     5281 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     5341 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     5401 ttatgcattt ctttccagac tt
//
