LOCUS       sequence-486673-        6006 bp    DNA     circular SYN 18-SEP-2025
DEFINITION  Phosphatase from E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6006)
  AUTHORS   Hoffpauir ZA, Lamb AL
  TITLE     Identification of the phosphatase essential for riboflavin 
            biosynthesis in Aquifex aeolicus.
  JOURNAL   J Biol Chem. 2025 May;301(5):108443. doi: 10.1016/j.jbc.2025.108443.
            Epub 2025 Mar 25.
  PUBMED    40147773
REFERENCE   2  (bases 1 to 6006)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem."; date: "2025-05"; volume: "301(5):108443. doi"; pages: " 
            10.1016/j.jbc.2025.108443. Epub 2025 Mar 25"
COMMENT     Sequence Label: YigB
FEATURES             Location/Qualifiers
     source          1..6006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(884..901)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(911..928)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             947..969
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    984..1008
                     /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     complement(1008..1027)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1009..1027)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1111..1130)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1336..1413
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1414..2496
                     /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(1433..1452)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3305..3496
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3490..3512)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3598..3737
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3653..3672
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3752..3769)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3923..4511)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4003..4022)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4622..4641)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4633..5448
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4633..5448
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4706..4725)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5540..5995)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5677..5698)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5889..5908
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagttagata aggctggtca
      181 ggctcgccaa acggctgatt tccaggtgcg gcagcaggcg gctatcccag gtttgcatta
      241 agtcgccgtt ctctgggcga atccagcagg cttgcatacc cgagcggatc gcaccgccaa
      301 catcggtcgt caagtcgtcg cccacgtgca ggatttcacc aataggcacg ttcagctttt
      361 cagccgccag gaagtacata tcgctaaacg gtttggaacg gccgtgcgga cctgcgcgca
      421 gcacgaactc gaaatagtca cccagaccaa acagctccgg ctgggcatta ccgttggtga
      481 ttgcaaccag cggccatttc ttcgccagct gtttcagggt gtcatgggtc tgctgcggga
      541 catcaatacg agaacgccac ttagcaaagt taatcatggc tgcatgcgcg cctgcgctcg
      601 cttcctccgc ggacaggcca gcgtccagca tggcttgttc gatcgaacga aaacgccaac
      661 gggtgacgtc gtggtagatc tccggctccg cctcacgcac agcctggcgc agacgttgga
      721 gatcttcgtt ttgaaaagat ctcaatgccg gatggtaatt ctgaacgaac gtcaacgctt
      781 cacgctcggt gcgcagaata accggacgat tatcatacag agtatcgtcg agatcgaacg
      841 tcaacgcact tattcgtcct aggggtctat aaaacctcat atggctgccg cgcggcacca
      901 ggccgctgct gtgatgatga tgatgatggc tgctgcccat ggtatatctc cttcttaaag
      961 ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt
     1021 cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca
     1081 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     1141 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
     1201 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
     1261 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
     1321 agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
     1381 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     1441 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     1501 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     1561 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     1621 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     1681 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     1741 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     1801 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     1861 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     1921 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     1981 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     2041 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     2101 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     2161 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     2221 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     2281 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     2341 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     2401 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     2461 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     2521 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag
     2581 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     2641 catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt
     2701 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct
     2761 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat
     2821 cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     2881 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     2941 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     3001 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     3061 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     3121 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     3181 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     3241 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     3301 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     3361 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     3421 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     3481 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     3541 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     3601 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     3661 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     3721 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3781 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3841 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3901 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3961 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4021 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4081 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4141 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4201 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4261 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4321 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4381 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4441 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4501 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4561 actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat
     4621 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat
     4681 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     4741 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     4801 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     4861 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     4921 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca
     4981 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     5041 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     5101 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     5161 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     5221 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     5281 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     5341 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     5401 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat
     5461 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5521 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5581 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     5641 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5701 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5761 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5821 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5881 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5941 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     6001 tcgcca
//
