LOCUS       sequence-468266-        6568 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Gateway entry vector for an inducible sg09 resistant PCSK5_T288P 
            mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6568)
  TITLE     Janes Lab Plasmids 2025
REFERENCE   2  (bases 1 to 6568)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pEN_TT PCSK5(sgRR,T288P)-V5
FEATURES             Location/Qualifiers
     source          1..6568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..48
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    67..166
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        198..512
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    206..224
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    242..260
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    277..295
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    313..331
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    349..367
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    384..402
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    420..438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     485..509
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             3258..3299
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(3338..3437)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3549..3568)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3627..3646)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(4220..4753)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4942..4947,4948..4964,4965..4970))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      5627..6215
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6116..6135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      6380..6466
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(6558..6568,1..17)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgacggatg gcctttttgc gtttctacaa actcttcctg ttagttagtt acttaagctc
       61 gggccccaaa taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat
      121 aagcaatgct tttttataat gccaactttg tacaaaaaag caggctttaa aggaaccaat
      181 tcagtcgact ggatcccgag tttactccct atcagtgata gagaacgtat gtcgagttta
      241 ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat agagaacgta
      301 tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg
      361 atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt cgagtttact
      421 ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag gcctatataa
      481 gcagagctcg tttagtgaac cgtcagatcg ccactagtat gggctggggg agccgctgct
      541 gctgcccggg acgtttggac ctgctgtgcg tgctggcgct gctcgggggc tgcctgctcc
      601 ccgtgtgtcg gacgcgcgtc tacaccaacc actgggcagt caaaatcgcc gggggcttcc
      661 cggaggccaa ccgtatcgcc agcaagtacg gattcatcaa cataggacag ataggggccc
      721 tgaaggacta ctaccacttc taccatagca ggacgattaa aaggtcagtt atctcgagca
      781 gagggaccca cagtttcatt tcaatggaac caaaggtgga atggatccaa cagcaagtgg
      841 taaaaaagcg gacaaagagg gattatgact tcagtcgtgc ccagtctacc tatttcaatg
      901 atcccaagtg gcccagcatg tggtatatgc actgcagtga caatacacat ccctgccagt
      961 ctgacatgaa tatcgaagga gcctggaaga gaggctacac ggggaagaac atcgtcgtca
     1021 ctatcctgga tgacggaatt gagagaaccc atccagatct gatgcaaaac tacgatgctc
     1081 tggcaagttg cgacgtgaat gggaatgact tggacccaat gcctcgttat gatgcaagca
     1141 acgagaacaa gcatgggact cgctgtgctg gagaagtggc agccgctgca aacaattcgc
     1201 actgcacagt cggaattgct ttcaacgcca agatcggagg agtgcgaatg ctggacggag
     1261 atgtcacgga catggttgaa gcaaaatcag ttagcttcaa cccccagcac gtgcacattt
     1321 acagcgccag ctggggcccg gatgatgatg gcaagactgt ggacggacca gcccccctcc
     1381 cccggcaagc ctttgaaaac ggcgttagaa tggggcggag aggcctcggc tctgtgtttg
     1441 tttgggcatc tggaaatggt ggaaggagca aagaccactg ctcctgtgat ggctacacca
     1501 acagcatcta caccatctcc atcagcagca ctgcagaaag cggaaagaaa ccttggtacc
     1561 tggaagagtg ttcatccacg ctggccacaa cctacagcag cggggagtcc tacgataaga
     1621 aaatcatcac tacagatctg aggcagcgtt gcacggacaa ccacactggg acgtcagcct
     1681 cagcccccat ggctgcaggc atcattgcgc tggccctgga agccaatccg tttctgacct
     1741 ggagagacgt acagcatgtt attgtcagga cttcccgtgc gggacatttg aacgctaatg
     1801 actggaaaac caatgctgct ggttttaagg tgagccatct ttatggattt ggactgatgg
     1861 acgcagaagc catggtgatg gaggcagaga agtggaccac cgttccccgg cagcacgtgt
     1921 gtgtggagag cacagaccga caaatcaaga caatccgccc taacagtgca gtgcgctcca
     1981 tctacaaagc ttcaggctgc tcggataacc ccaaccgcca tgtcaactac ctggagcacg
     2041 tcgttgtgcg catcaccatc acccacccca ggagaggaga cctggccatc tacctgacct
     2101 cgccctctgg aactaggtct cagcttttgg ccaacaggct atttgatcac tccatggaag
     2161 gattcaaaaa ctgggagttc atgaccattc attgctgggg agaaagagct gctggtgact
     2221 gggtccttga agtttatgat actccctctc agctaaggaa ctttaagact ccaggtaaat
     2281 tgaaagaatg gtctttggtc ctctacggca cctccgtgca gccatattca ccaaccaatg
     2341 aatttccgaa agtggaacgg ttccgctata gccgagttga agaccccaca gacgactatg
     2401 gcacagagga ttatgcaggt ccctgcgacc ctgagtgcag tgaggttggc tgtgacgggc
     2461 caggaccaga ccactgcaat gactgtttgc actactacta caagctgaaa aacaatacca
     2521 ggatctgtgt ctccagctgc ccccctggcc actaccacgc cgacaagaag cgctgcagga
     2581 agtgtgcccc caactgtgag tcctgctttg ggagccatgg tgaccaatgc atgtcctgca
     2641 aatatggata ctttctgaat gaagaaacca acagctgtgt tactcactgc cctgatgggt
     2701 catatcagga taccaagaaa aatctttgcc ggaaatgcag tgaaaactgc aagacatgta
     2761 ctgaattcca taactgtaca gaatgtaggg atgggttaag cctgcaggga tcccggtgct
     2821 ctgtctcctg tgaagatgga cggtatttca acggccagga ctgccagccc tgccaccgct
     2881 tctgcgccac ttgtgctggg gcaggagctg atgggtgcat taactgcaca gagggctact
     2941 tcatggagga tgggagatgc gtgcagagct gtagtatcag ctattacttt gaccactctt
     3001 cagagaatgg atacaaatcc tgcaaaaaat gtgatatcag ttgtttgacg tgcaatggcc
     3061 caggattcaa gaactgtaca agctgcccta gtgggtatct cttagactta ggaatgtgtc
     3121 aaatgggagc catttgcaag gatgcaacgg aagagtcctg ggcggaagga ggcttctgta
     3181 tgcttgtgaa aaagaacaat ctgtgccaac ggaaggttct tcaacaactt tgctgcaaaa
     3241 catgtacatt tcaaggcggt aagcctatcc ctaaccctct cctcggtctc gattctacgt
     3301 aacaattgga agactaatgc gttttcgaga tatctagacc cagctttctt gtacaaagtt
     3361 ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa
     3421 aataaaatca ttatttgcca tccagctgca gctctggccc gtgtctcaaa atctctgatg
     3481 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     3541 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     3601 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     3661 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     3721 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     3781 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     3841 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     3901 aggtgaaaat attgttgatg cgctggcagt gtccctgcgc cggttgcatt cgattcctgt
     3961 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     4021 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     4081 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     4141 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaggcc gggaagccga
     4201 tctcggcttg aacgaattgt taggtggcgg tacttgggtc gatatcaaag tgcatcactt
     4261 cttcccgtat gcccaacttt gtatagagag ccactgcggg atcgtcaccg taatctgctt
     4321 gcacgtagat cacataagca ccaagcgcgt tggcctcatg cttgaggaga ttgatgagcg
     4381 cggtggcaat gccctgcctc cggtgctcgc cggagactgc gagatcatag atatagatct
     4441 cactacgcgg ctgctcaaac ttgggcagaa cgtaagccgc gagagcgcca acaaccgctt
     4501 cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg gagcaagttc ccgaggtaat
     4561 cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga ccgaaaagat
     4621 caagagcagc ccgcatggat ttgacttggt cagggccgag cctacatgtg cgaatgatgc
     4681 ccatacttga gccacctaac tttgttttag ggcgactgcc ctgctgcgta acatcgttgc
     4741 tgctgcgtaa catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct
     4801 tgctgcttgg atgcccgagg catagactgt acaaaaaaac agtcataaca agccatgaaa
     4861 accgccactg cgccgttacc accgctgcgt tcggtcaagg ttctggacca gttgcgtgag
     4921 cgcatacgct acttgcatta cagtttacga accgaacagg cttatgtcaa ctgggttcgt
     4981 gccttcatcc gtttccacgg tgtgcgtcac ccggcaacct tgggcagcag cgaagtcgag
     5041 gcatttctgt cctggctggc gaacgagcgc aaggtttcgg tctccacgca tcgtcaggca
     5101 ttggcggcct tgctgttctt ctacggcaag gtgctgtgca cggatctgcc ctggcttcag
     5161 gagatcggaa gacctcggcc gtcgcggcgc ttgccggtgg tgctgacccc ggatgaagtg
     5221 gttcgcatcc tcggttttct ggaaggcgag catcgtttgt tcgcccagga ctctagctat
     5281 agttctagtg gttggctact aataggttgt attgatgttg gacgagtcgg aatcgcagac
     5341 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     5401 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     5461 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     5521 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagtttt
     5581 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5641 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5701 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5761 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5821 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5881 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5941 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6001 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6061 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6121 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6181 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6241 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6301 ctgtggataa ccgtattacc gctagcatgg atctcgggga cgtctaacta ctaagcgaga
     6361 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcggaagact gggcctttcg
     6421 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     6481 tttgaacgtt gtgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     6541 caggcatcaa actaagcaga aggccatc
//
