LOCUS       sequence-458996-        7388 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For T-REX expriments of PCSK1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7388)
  TITLE     Organ-specific electrophile responsivity mapping in live C. elegans
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7388)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCS2+8-PCSK1-flag-tev-halo-his
FEATURES             Location/Qualifiers
     source          1..7388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2398..2421
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2428..2448
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2455..3345
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             3355..3372
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    3440..3574
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3464..3483)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3518..3537
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3687..3707)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3687..3705)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3726..3742)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3726..3742)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3739..3761)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3750..3766
                     /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)."
     promoter        complement(join(3774..3780,3781..3798,3799..3804))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3819..3840
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3957..3974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4128..4716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4208..4227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4887..5678,5679..5747))
                     /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     5510..5529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5748..5852)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5878..6333)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6015..6036)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6227..6246
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6404..7388
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atccactagt ggcgcgccat taattaaagg ccggccagcg
      121 atcgccggac ccaccatgga gcgaagagcc tggagtctgc agtgcactgc tttcgtcctc
      181 ttttgcgctt ggtgtgcact gaacagtgca aaagcgaaaa ggcaatttgt caatgaatgg
      241 gcagcggaga tccccggggg cccggaagca gcctcggcca tcgccgagga gctgggctat
      301 gaccttttgg gtcagattgg ttcacttgaa aatcactact tattcaaaca taaaaaccac
      361 cccagaaggt ctcgaaggag tgcctttcat atcactaaga gattatctga tgatgatcgt
      421 gtgatatggg ctgaacaaca gtatgaaaaa gaaagaagta aacgttcagc tctaagggac
      481 tcagcactaa atctcttcaa tgatcccatg tggaatcagc aatggtactt gcaagatacc
      541 aggatgacgg cagccctgcc caagctggac cttcatgtga tacctgtttg gcaaaaaggc
      601 attacgggca aaggagttgt tatcaccgta ctggatgatg gtttggagtg gaatcacacg
      661 gacatttatg ccaactatga tccagaggct agctatgatt ttaatgataa tgaccatgat
      721 ccatttcccc gatatgatcc cacaaacgag aacaaacacg ggaccagatg tgcaggagaa
      781 attgccatgc aagcaaataa tcacaaatgc ggggttggag ttgcatacaa ttccaaagtt
      841 ggaggcataa gaatgctgga tggcattgtg acggatgcta ttgaggccag ttcaattgga
      901 ttcaatcctg gacacgtgga tatttacagt gcaagctggg gccctaatga tgatgggaaa
      961 actgtggagg ggcctggccg gctagcccag aaggcttttg aatatggtgt caaacagggg
     1021 agacagggga aggggtccat cttcgtctgg gcttcgggaa acggggggcg tcagggagat
     1081 aattgtgact gtgatggcta cacagacagc atctacacca tctccatcag cagtgcctcc
     1141 cagcaaggcc tatccccctg gtacgctgag aagtgctcct ccacactggc cacctcttac
     1201 agcagcggag attacaccga ccagagaatc acgagcgctg acctgcacaa tgactgcacg
     1261 gagacgcaca caggcacctc ggcctctgca cctctggctg ctggcatctt cgctctggcc
     1321 ctggaagcaa acccaaatct cacctggcga gatatgcagc acctggttgt ctggacctct
     1381 gagtatgacc cgctggccaa taaccctgga tggaaaaaga atggagcagg cttgatggtg
     1441 aatagtcgat ttggatttgg cttgctaaat gccaaagctc tggtggattt agctgacccc
     1501 aggacctgga ggagcgtgcc tgagaagaaa gagtgtgttg taaaggacaa tgactttgag
     1561 cccagagccc tgaaagctaa tggagaagtt atcattgaaa ttccaacaag agcttgtgaa
     1621 ggacaagaaa atgctatcaa gtccctggag catgtacaat ttgaagcaac aattgaatat
     1681 tcccgaagag gagaccttca tgtcacactt acttctgctg ctggaactag cactgtgctc
     1741 ttggctgaaa gagaacggga tacatctcct aatggcttta agaattggga cttcatgtct
     1801 gttcacacat ggggagagaa ccctataggt acttggactt tgagaattac agacatgtct
     1861 ggaagaattc aaaatgaagg aagaattgtg aactggaagc tgattttgca cgggacctct
     1921 tctcagccag agcatatgaa gcagcctcgt gtgtacacgt cctacaacac tgttcagaat
     1981 gacagaagag gggtggagaa gatggtggat ccaggggagg agcagcccac acaagagaac
     2041 cctaaggaga acaccctggt gtccaaaagc cccagcagca gcagcgtagg gggccggagg
     2101 gatgagttgg aggagggagc cccttccgag gccatgctgc gactcctgca aagtgctttc
     2161 agtaaaaact caccgccaaa gcaatcacca aagaagtccc caactgcaaa gctcaacatc
     2221 ccttatgaaa acttctacga agccctggaa aagctgaaca aaccttccca gcttaaagac
     2281 tctgaagaca gtctgtataa tgactatgtt gatgtttttt ataacactaa accttacaag
     2341 cacagagacg accggctgct tcaagctctg gtggacattc tgaatgagga aaatatggat
     2401 tataaagacg atgacgacaa atcaggggaa aacttgtatt tccagggctc agggatggca
     2461 gaaatcggta ctggctttcc attcgacccc cattatgtgg aagtcctggg cgagcgcatg
     2521 cactacgtcg atgttggtcc gcgcgatggc acccctgtgc tgttcctgca cggtaacccg
     2581 acctcctcct acgtgtggcg caacatcatc ccgcatgttg caccgaccca tcgctgcatt
     2641 gctccagacc tgatcggtat gggcaaatcc gacaaaccag acctgggtta tttcttcgac
     2701 gaccacgtcc gcttcatgga tgccttcatc gaagccctgg gtctggaaga ggtcgtcctg
     2761 gtcattcacg actggggctc cgctctgggt ttccactggg ccaagcgcaa tccagagcgc
     2821 gtcaaaggta ttgcatttat ggagttcatc cgccctatcc cgacctggga cgaatggcca
     2881 gaatttgccc gcgagacctt ccaggccttc cgcaccaccg acgtcggccg caagctgatc
     2941 atcgatcaga acgtttttat cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact
     3001 gaagtcgaga tggaccatta ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg
     3061 tggcgcttcc caaacgagct gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc
     3121 gaagaataca tggactggct gcaccagtcc cctgtcccga agctgctgtt ctggggcacc
     3181 ccaggcgttc tgatcccacc ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc
     3241 aaggctgtgg acatcggccc gggtctgaat ctgctgcaag aagacaaccc ggacctgatc
     3301 ggcagcgaga tcgcgcgctg gctgtcgacg ctcgagattt ccggcggcag cagccatcat
     3361 catcatcatc atgggtcagg gtaataatcg aattcaaggc ctctcgagcc tctagaacta
     3421 tagtgagtcg tattacgtag atccagacat gataagatac attgatgagt ttggacaaac
     3481 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3541 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     3601 gtttcaggtt cagggggagg tgtgggaggt tttttaattc gcggccgcgg cgccaatgca
     3661 ttgggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     3721 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3781 cgagccggga gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3841 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3901 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3961 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4021 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4081 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4141 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4201 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4261 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4321 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4381 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4441 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4501 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4561 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4621 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4681 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4741 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4801 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4861 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4921 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4981 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5041 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5101 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5161 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5221 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5281 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5341 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5401 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5461 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5521 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5581 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5641 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5701 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5761 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5821 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     5881 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5941 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     6001 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     6061 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     6121 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     6181 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     6241 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     6301 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     6361 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagtcgacc gccaattcaa
     6421 tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt gccaattcaa
     6481 tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca gccaattcaa
     6541 tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt gccaactggg
     6601 gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg tgccaagtcc
     6661 gccatattga attggcatgg tgccaataat ggcggccata ttggctatat gccaggatca
     6721 atatataggc aatatccaat atggccctat gccaatatgg ctattggcca ggttcaatac
     6781 tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt gacgtagata
     6841 gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac cgcccatagc
     6901 cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac gtcatatggg
     6961 cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta aatggcccgc
     7021 ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa tgggatggct
     7081 cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg taaatggccc
     7141 acttggcagt acatcaatat ctattaatag taacttggca agtacattac tattggaagt
     7201 acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg gcccgcgatg
     7261 gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa atgggcgttc
     7321 cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc aatgctcgtt
     7381 tagggaac
//
