LOCUS       sequence-263135-        6270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of recombinant Arabidopsis thaliana 
            ferrochelatase1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6270)
  AUTHORS   Shimizu T, Kacprzak SM, Mochizuki N, Nagatani A, Watanabe S, Shimada
            T, Tanaka K, Hayashi Y, Arai M, Leister D, Okamoto H, Terry MJ, 
            Masuda T
  TITLE     The retrograde signaling protein GUN1 regulates tetrapyrrole 
            biosynthesis.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Dec 3;116(49):24900-24906. doi: 
            10.1073/pnas.1911251116. Epub 2019 Nov 15.
  PUBMED    31732672
REFERENCE   2  (bases 1 to 6270)
  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: 
            "10.1073/pnas.1911251116"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2019-12-3- 3"; volume: "116"; issue: "49"; pages: 
            "24900-24906"
COMMENT     Sequence Label: pGEX2T AtFC1
FEATURES             Location/Qualifiers
     source          1..6270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     RBS             1682..1690
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(2320..2342)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2487..2505)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2573..2677
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2678..2746,2747..3538)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2896..2915)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3709..4297
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4198..4217
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4451..4468
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4541..4618
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4619..5701
                     /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(4638..4657)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5714..5735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5750..5755,5756..5773,5774..5780)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5788..5804
                     /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)."
     primer_bind     5812..5828
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5812..5828
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5824..6270,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5844..5861)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5844..5860)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5853..5875)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatcctctca gagaaattct ctgtctttga
      961 ttcaatgcga tataaaagag agatctttcg gagagtctat gacgatcacg aatcgtggat
     1021 tgagttttaa gacgaatgtg tttgagcaag ctcgttctgt gactggagac tgttcttatg
     1081 atgaaacttc agctaaagca cgttctcatg ttgttgcaga agataagatt ggtgtcttgc
     1141 ttttgaattt aggtggtcct gaaactctta acgatgttca acctttcttg tataatctct
     1201 ttgctgatcc ggatattata aggcttccta gaccatttca gtttcttcaa gggactatag
     1261 ctaagtttat atctgttgtt cgtgctccga aatctaaaga agggtatgct gctattggtg
     1321 gtggctctcc tttgcgtaaa ataactgatg agcaagcgga tgctattaag atgtctttgc
     1381 aagcgaagaa cattgctgct aatgtctatg ttggtatgcg gtattggtat ccgttcactg
     1441 aggaggctgt tcagcagata aagaaggaca aaattactag acttgttgta ctgccattgt
     1501 atcctcagta ttctatctct acaacgggtt caagcatacg cgttctccaa gatttattca
     1561 ggaaagatcc gtacctagct ggagtgccgg tagctattat aaagtcctgg taccaaaggc
     1621 gaggctatgt caattctatg gctgacctca ttgagaagga gcttcaaact ttctctgatc
     1681 ctaaggaggt tatgatattc ttcagtgccc atggtgttcc ggtcagctac gtagagaatg
     1741 ctggagatcc gtaccagaag cagatggaag agtgtattga cttgataatg gaagagctaa
     1801 aagccagagg ggttcttaac gaccataaat tggcatacca gagtcgtgtt ggccctgttc
     1861 aatggctgaa gccatacacc gatgaggttc ttgtcgacct tggtaagagt ggtgttaaga
     1921 gtctactagc cgttccagtc agtttcgtga gtgagcacat tgagacactt gaggagatag
     1981 acatggagta tagggaatta gctcttgagt caggggtaga gaactgggga cgggtacccg
     2041 cgctaggtct cacaccatcc ttcatcaccg acttagctga tgcagtgata gaatcacttc
     2101 cttcagcaga agcaatgtca aacccaaatg cagtggttga ctcagaagat agcgagtcgt
     2161 cagatgcttt cagttacatt gtcaagatgt tcttcggttc gattctggct ttcgtcctac
     2221 ttctctcccc aaagatgttc catgcgttcc ggaacctata ggaattcatc gtgactgact
     2281 gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     2341 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     2401 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     2461 agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2521 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2581 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2641 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2701 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2761 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2821 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2881 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     2941 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3001 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3061 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3121 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3181 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     3241 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3301 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3361 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3421 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3481 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3541 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3601 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3661 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3721 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3781 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3841 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3901 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3961 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4021 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4081 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4141 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4201 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4261 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4321 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4381 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4441 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     4501 ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg aatggtgcaa
     4561 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     4621 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     4681 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     4741 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     4801 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     4861 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     4921 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     4981 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     5041 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     5101 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     5161 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     5221 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     5281 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     5341 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     5401 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     5461 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     5521 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     5581 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     5641 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5701 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     5761 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     5821 gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     5881 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     5941 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     6001 cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt
     6061 cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta cgatgcgccc
     6121 atctacacca acgtaaccta tcccattacg gtcaatccgc cgtttgttcc cacggagaat
     6181 ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca ggaaggccag
     6241 acgcgaatta tttttgatgg cgttggaatt
//
