LOCUS       sequence-183823-        6211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PCY1:S495A from Saponaria vaccaria possesing a N-terminal 
            His6-tag (TEV cleavable) in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6211)
  AUTHORS   Ludewig H, Czekster CM, Oueis E, Munday ES, Arshad M, Synowsky SA, 
            Bent AF, Naismith JH
  TITLE     Characterization of the Fast and Promiscuous Macrocyclase from Plant
            PCY1 Enables the Use of Simple Substrates.
  JOURNAL   ACS Chem Biol. 2018 Mar 16;13(3):801-811. doi: 
            10.1021/acschembio.8b00050. Epub 2018 Feb 12.
  PUBMED    29377663
REFERENCE   2  (bases 1 to 6211)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acschembio.8b00050"; journalName: "ACS Chem Biol"; date: 
            "2018-03-16- 16"; volume: "13"; issue: "3"; pages: "801-811"
COMMENT     Sequence Label: pJ414-PCY1:S495A
FEATURES             Location/Qualifiers
     source          1..6211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     CDS             89..106
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             107..127
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      2316..2363
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2586..2605)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2611..2630)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2672..2763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2764..2832,2833..3624)
                     /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     complement(2982..3001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             3707..4789
                     /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     complement(3726..3745)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      4875..5463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5364..5383
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5617..5634
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5732..5749
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5733..5749
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt ttaggaggta aacatatgca ccatcatcat caccacgaga atttgtattt
      121 ccaaggtatg gctaccagcg gtttcagcaa gccgctgcat tatccgccgg tccgccgcga
      181 tgaaaccgtt gttgacgatt actttggtgt gaaagtcgcg gacccatacc gctggttgga
      241 ggacccgaac tctgaggaaa cgaaagaatt tgttgataac caagaaaaac tggcaaactc
      301 ggtcctggaa gagtgtgagc tgattgacaa gtttaagcaa aagattatcg atttcgtgaa
      361 tttcccgcgt tgcggtgtgc cgttccgccg cgccaacaaa tactttcact tctacaacag
      421 cggcctgcag gcgcaaaacg tctttcagat gcaagatgat ttggacggta agcctgaagt
      481 tctgtacgat ccgaatctgc gcgagggcgg tcgcagcggc ctgtccttgt acagcgtcag
      541 cgaagatgcc aagtactttg cctttggtat tcatagcggc ctgaccgaat gggtgaccat
      601 caagattctg aaaactgaag atcgttctta tttgccggat accctggaat gggtgaagtt
      661 cagcccggct atctggaccc acgataacaa aggtttcttt tactgtccgt atccgccact
      721 gaaagagggc gaggaccaca tgactcgctc cgcagttaac caagaggctc gttatcactt
      781 cctgggcacg gatcagagcg aagatatcct gctgtggcgc gatctggaga atccggcgca
      841 ccacctgaaa tgtcagatca ccgacgatgg caagtatttt ctgctgtata tcttggacgg
      901 ttgtgatgat gccaataaag tttactgctt ggatctgacg aagctgccga acggcctgga
      961 gagctttcgt ggtcgcgaag attcagcacc gttcatgaag ctgattgata gcttcgacgc
     1021 gtcgtacacg gcgattgcta atgatggttc cgtgttcacc ttccagacga acaaagacgc
     1081 accgcgtaag aaactggttc gcgtggatct gaataatccg agcgtttgga cggacctggt
     1141 gcctgagagc aaaaaagatc tgctggagag cgcccacgca gtgaatgaaa atcaactgat
     1201 tctgcgttac ttgagcgatg tgaaacatgt tctggaaatt cgtgacctgg agagcggtgc
     1261 actgcaacac cgtctgccga tcgacattgg cagcgttgac ggtattacgg cgcgtcgtcg
     1321 cgactccgtg gttttcttta agttcaccag catcctgacc ccgggcattg tttatcagtg
     1381 cgacctgaaa aacgacccga cccagctgaa gatcttccgt gagagcgtgg tcccggactt
     1441 cgaccgctcc gaatttgaag tcaaacaagt tttcgttccg agcaaggacg gtaccaaaat
     1501 cccgatcttc attgccgcgc gtaagggcat cagcttggac ggcagccacc cgtgcgagat
     1561 gcacggttat ggcggttttg gcattaatat gatgccaacg ttcagcgcag cgcgtattgt
     1621 ctttctcaag catctgggtg gcgtcttttg cctggcgaat atccgtggcg gcggcgaata
     1681 cggtgaggaa tggcacaagg ccggctttcg tgacaaaaag caaaatgttt ttgacgattt
     1741 catctcggcg gcggagtatc tgatcagcag cggttatacc aaggcacgtc gtgtggcgat
     1801 cgagggtggt agcaatggtg gcctgctggt cgcggcgtgt attaaccaac gcccggactt
     1861 gtttggttgc gctgaggcca actgcggtgt catggatatg ctgcgttttc ataagttcac
     1921 cctgggttac ctgtggactg gtgattacgg ttgtagcgat aaggaagaag aattcaagtg
     1981 gctgattaaa tactctccaa ttcacaacgt gcgtcgtccg tgggagcagc cgggtaacga
     2041 agaaacgcaa tatcctgcga ccatgattct gaccgcggac cacgacgacc gcgtagtccc
     2101 gctgcactct ttcaaactgc tggcgaccat gcaacacgtg ctgtgcacta gcttagagga
     2161 ctctccgcag aagaatccga ttatcgcacg tattcagcgt aaagctgccc attacggtcg
     2221 tgcgacgatg acgcagatcg cagaagtggc ggaccgctac ggtttcatgg cgaaagccct
     2281 ggaagcaccg tggatcgatt gatgaaagct tccccctagc ataacccctt ggggcctcta
     2341 aacgggtctt gaggggtttt ttgcccctga gacgcgtcaa tcgagttcgt acctaagggc
     2401 gacaccccat aattagcccg ggcgaaaggc ccagtctttc gactgagcct ttcgttttat
     2461 ttgatgcctg gcagttccct actctcgcat ggggagtccc cacactacca tcggcgctac
     2521 ggcgtttcac ttctgagttc ggcatggggt caggtgggac caccgcgcta ctgccgccag
     2581 gcaaacaagg ggtgttatga gccatattca ggtataaatg ggctcgcgat aatgttcaga
     2641 attggttaat tggttgtaac actgacccct atttgtttat ttttctaaat acattcaaat
     2701 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2761 aatatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2821 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2881 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2941 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3001 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3061 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3121 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3181 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3241 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3301 atgcctgtag cgatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3361 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3421 cgctcggccc ttccggctgg ctggtttatt gctgataaat ccggagccgg tgagcgtggt
     3481 tctcgcggta tcatcgcagc gctggggcca gatggtaagc cctcccgtat cgtagttatc
     3541 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3601 gcctcactga ttaagcattg gtaagcggcg cgccatcgaa tggcgcaaaa cctttcgcgg
     3661 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatatga aaccagtaac
     3721 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     3781 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     3841 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     3901 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     3961 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     4021 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     4081 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     4141 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgagga
     4201 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     4261 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     4321 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     4381 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     4441 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     4501 tgcggatatc tcggtagtgg gatacgacga taccgaagat agctcatgtt atatcccgcc
     4561 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     4621 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgccagtct cactggtgaa
     4681 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4741 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgac tcatgaccaa
     4801 aatcccttaa cgtgagttac gcgcgcgtcg ttccactgag cgtcagaccc cgtagaaaag
     4861 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4921 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4981 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5041 ttagcccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5101 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5161 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5221 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5281 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5341 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5401 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5461 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5521 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5581 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5641 gaaggcgaga gtagggaact gccaggcatc aaactaagca gaaggcccct gacggatggc
     5701 ctttttgcgt ttctacaaac tctttctgtg ttgtaaaacg acggccagtc ttaagctcgg
     5761 gccccctggg cggttctgat aacgagtaat cgttaatccg caaataacgt aaaaacccgc
     5821 ttcggcgggt ttttttatgg ggggagttta gggaaagagc atttgtcaga atatttaagg
     5881 gcgcctgtca ctttgcttga tatatgagaa ttatttaacc ttataaatga gaaaaaagca
     5941 acgcacttta aataagatac gttgcttttt cgattgatga acacctataa ttaaactatt
     6001 catctattat ttatgatttt ttgtatatac aatatttcta gtttgttaaa gagaattaag
     6061 aaaataaatc tcgaaaataa taaagggaaa atcagttttt gatatcaaaa ttatacatgt
     6121 caacgataat acaaaatata atacaaacta taagatgtta tcagtattta ttatcattta
     6181 gaataaattt tgtgtcgccc ttccgcgaaa t
//
