LOCUS       sequence-189892-        6211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PCY1:H695Q 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:H695Q
FEATURES             Location/Qualifiers
     source          1..6211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             19..39
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      2228..2275
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2498..2517)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2523..2542)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2584..2675
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2676..2744,2745..3536)
                     /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(2894..2913)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             3619..4701
                     /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(3638..3657)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      4787..5375
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5276..5295
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5529..5546
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5644..5661
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5645..5661
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6124..6143
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6124..6142
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6143..6167
                     /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)."
ORIGIN
        1 caccatcatc atcaccacga gaatttgtat ttccaaggta tggctaccag cggtttcagc
       61 aagccgctgc attatccgcc ggtccgccgc gatgaaaccg ttgttgacga ttactttggt
      121 gtgaaagtcg cggacccata ccgctggttg gaggacccga actctgagga aacgaaagaa
      181 tttgttgata accaagaaaa actggcaaac tcggtcctgg aagagtgtga gctgattgac
      241 aagtttaagc aaaagattat cgatttcgtg aatttcccgc gttgcggtgt gccgttccgc
      301 cgcgccaaca aatactttca cttctacaac agcggcctgc aggcgcaaaa cgtctttcag
      361 atgcaagatg atttggacgg taagcctgaa gttctgtacg atccgaatct gcgcgagggc
      421 ggtcgcagcg gcctgtcctt gtacagcgtc agcgaagatg ccaagtactt tgcctttggt
      481 attcatagcg gcctgaccga atgggtgacc atcaagattc tgaaaactga agatcgttct
      541 tatttgccgg ataccctgga atgggtgaag ttcagcccgg ctatctggac ccacgataac
      601 aaaggtttct tttactgtcc gtatccgcca ctgaaagagg gcgaggacca catgactcgc
      661 tccgcagtta accaagaggc tcgttatcac ttcctgggca cggatcagag cgaagatatc
      721 ctgctgtggc gcgatctgga gaatccggcg caccacctga aatgtcagat caccgacgat
      781 ggcaagtatt ttctgctgta tatcttggac ggttgtgatg atgccaataa agtttactgc
      841 ttggatctga cgaagctgcc gaacggcctg gagagctttc gtggtcgcga agattcagca
      901 ccgttcatga agctgattga tagcttcgac gcgtcgtaca cggcgattgc taatgatggt
      961 tccgtgttca ccttccagac gaacaaagac gcaccgcgta agaaactggt tcgcgtggat
     1021 ctgaataatc cgagcgtttg gacggacctg gtgcctgaga gcaaaaaaga tctgctggag
     1081 agcgcccacg cagtgaatga aaatcaactg attctgcgtt acttgagcga tgtgaaacat
     1141 gttctggaaa ttcgtgacct ggagagcggt gcactgcaac accgtctgcc gatcgacatt
     1201 ggcagcgttg acggtattac ggcgcgtcgt cgcgactccg tggttttctt taagttcacc
     1261 agcatcctga ccccgggcat tgtttatcag tgcgacctga aaaacgaccc gacccagctg
     1321 aagatcttcc gtgagagcgt ggtcccggac ttcgaccgct ccgaatttga agtcaaacaa
     1381 gttttcgttc cgagcaagga cggtaccaaa atcccgatct tcattgccgc gcgtaagggc
     1441 atcagcttgg acggcagcca cccgtgcgag atgcacggtt atggcggttt tggcattaat
     1501 atgatgccaa cgttcagcgc aagccgtatt gtctttctca agcatctggg tggcgtcttt
     1561 tgcctggcga atatccgtgg cggcggcgaa tacggtgagg aatggcacaa ggccggcttt
     1621 cgtgacaaaa agcaaaatgt ttttgacgat ttcatctcgg cggcggagta tctgatcagc
     1681 agcggttata ccaaggcacg tcgtgtggcg atcgagggtg gtagcaatgg tggcctgctg
     1741 gtcgcggcgt gtattaacca acgcccggac ttgtttggtt gcgctgaggc caactgcggt
     1801 gtcatggata tgctgcgttt tcataagttc accctgggtt acctgtggac tggtgattac
     1861 ggttgtagcg ataaggaaga agaattcaag tggctgatta aatactctcc aattcacaac
     1921 gtgcgtcgtc cgtgggagca gccgggtaac gaagaaacgc aatatcctgc gaccatgatt
     1981 ctgaccgcgg accacgacga ccgcgtagtc ccgctgcact ctttcaaact gctggcgacc
     2041 atgcaacacg tgctgtgcac tagcttagag gactctccgc agaagaatcc gattatcgca
     2101 cgtattcagc gtaaagctgc ccagtacggt cgtgcgacga tgacgcagat cgcagaagtg
     2161 gcggaccgct acggtttcat ggcgaaagcc ctggaagcac cgtggatcga ttgatgaaag
     2221 cttcccccta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgcccct
     2281 gagacgcgtc aatcgagttc gtacctaagg gcgacacccc ataattagcc cgggcgaaag
     2341 gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
     2401 atggggagtc cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg
     2461 gtcaggtggg accaccgcgc tactgccgcc aggcaaacaa ggggtgttat gagccatatt
     2521 caggtataaa tgggctcgcg ataatgttca gaattggtta attggttgta acactgaccc
     2581 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2641 gataaatgct tcaataatat tgaaaaagga agaatatgag tattcaacat ttccgtgtcg
     2701 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     2761 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     2821 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     2881 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     2941 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3001 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3061 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3121 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3181 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcgatggca acaacgttgc
     3241 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3301 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3361 ttgctgataa atccggagcc ggtgagcgtg gttctcgcgg tatcatcgca gcgctggggc
     3421 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3481 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaagcgg
     3541 cgcgccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     3601 caattcaggg tggtgaatat gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     3661 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     3721 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     3781 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     3841 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     3901 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     3961 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     4021 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     4081 cccatcaaca gtattatttt ctcccatgag gacggtacgc gactgggcgt ggagcatctg
     4141 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     4201 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     4261 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     4321 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     4381 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     4441 gataccgaag atagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     4501 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     4561 ggcaatcagc tgttgccagt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     4621 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4681 cgactggaaa gcgggcagtg actcatgacc aaaatccctt aacgtgagtt acgcgcgcgt
     4741 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4801 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4861 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4921 taccaaatac tgttcttcta gtgtagccgt agttagccca ccacttcaag aactctgtag
     4981 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5041 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5101 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5161 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5221 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5281 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5341 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5401 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5461 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5521 ccgagcgcag cgagtcagtg agcgaggaag cggaaggcga gagtagggaa ctgccaggca
     5581 tcaaactaag cagaaggccc ctgacggatg gcctttttgc gtttctacaa actctttctg
     5641 tgttgtaaaa cgacggccag tcttaagctc gggccccctg ggcggttctg ataacgagta
     5701 atcgttaatc cgcaaataac gtaaaaaccc gcttcggcgg gtttttttat ggggggagtt
     5761 tagggaaaga gcatttgtca gaatatttaa gggcgcctgt cactttgctt gatatatgag
     5821 aattatttaa ccttataaat gagaaaaaag caacgcactt taaataagat acgttgcttt
     5881 ttcgattgat gaacacctat aattaaacta ttcatctatt atttatgatt ttttgtatat
     5941 acaatatttc tagtttgtta aagagaatta agaaaataaa tctcgaaaat aataaaggga
     6001 aaatcagttt ttgatatcaa aattatacat gtcaacgata atacaaaata taatacaaac
     6061 tataagatgt tatcagtatt tattatcatt tagaataaat tttgtgtcgc ccttccgcga
     6121 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     6181 ttttgtttaa cttttaggag gtaaacatat g
//
