LOCUS       sequence-367935-        6293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CDS of L-HicDH codon optimized for expression in Synechocystis sp. 
            PCC 6803 in pET21a(+)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6293)
  AUTHORS   Jurkas V, Winkler CK, Poschenrieder S, Oliveira P, Pacheco CC, 
            Ferreira EA, Weissensteiner F, De Santis P, Kara S, Kourist R, 
            Tamagnini P, Kroutil W
  TITLE     Expression and activity of heterologous hydroxyisocaproate 
            dehydrogenases in Synechocystis sp. PCC 6803 DeltahoxYH.
  JOURNAL   Eng Microbiol. 2021 Nov 26;2(1):100008. doi: 
            10.1016/j.engmic.2021.100008. eCollection 2022 Mar.
  PUBMED    39628613
REFERENCE   2  (bases 1 to 6293)
  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"; journalName: "Eng 
            Microbiol."; date: "2021-11-26"; volume: "2(1):100008. doi"; pages: 
            " 10.1016/j.engmic.2021.100008. eCollection 2022 Mar"
COMMENT     Sequence Label: pET21a(+)_co_L-HicDH
FEATURES             Location/Qualifiers
     source          1..6293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..42
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(122..139)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(293..881)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(373..392)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1052..1843,1844..1912))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1675..1694
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1913..2017)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2043..2498)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2180..2201)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2392..2411
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2535..2582)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2578..2596
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2649..2666)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3610..3632
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3647..3671
                     /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(3671..3690)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3672..3690)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3774..3793)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3999..4076
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4077..5159
                     /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(4096..4115)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5968..6159
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6153..6175)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    join(6261..6293,1..107)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc
       61 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
      121 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
      181 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
      241 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
      301 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
      361 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
      421 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
      481 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
      541 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
      601 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
      661 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
      721 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
      781 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
      841 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
      901 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
      961 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     1021 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     1081 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     1141 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     1201 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     1261 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     1321 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg
     1381 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     1441 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     1501 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     1561 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     1621 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     1681 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     1741 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     1801 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     1861 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     1921 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     1981 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     2041 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     2101 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2161 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2221 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2281 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2341 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2401 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     2461 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccaa tccggatata
     2521 gttcctcctt tcagcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct
     2581 agttattgct cagcggtggc agcagccaac tcagcttcct ttcgggcttt gttagcagcc
     2641 ggatctcagt ggtggtggtg gtggtgctcg agcaaagtgt caacaatttc atcaaaccgc
     2701 tgctgaatgt agtccctaga ttgtagcagt ttttcttgtt cgtccgtcgt taagtctaaa
     2761 gttgtctcag ccagcacccc gtcacgcccg ataatggccg gataactcag gtacataccc
     2821 atatcgtcac gacgattact caccaccaat tcggcatgcg cgtcggccat caccgctttg
     2881 gcaattcgga tcgcgctggt cgctacacca tacgaagtgt agcccttccc gtttaagacg
     2941 gtgaagccac ctttccgagc ctcttcttca atagcagcca gatcgatatc accggcatcc
     3001 gccaaggtta cgataggttg ccccatgacc cggacagtac tccaggctac aaactggcta
     3061 ttgccgtgct cccccaagtt gtagcccgaa acagagcgag gatccaggtc aaaggcttca
     3121 cctacagctc tttgcatcct ggcagtatcg agcagtgtcc cggtaccaat caccttgtgg
     3181 gcgggaaagc ctgtcacgtg ttgaaaaagt gcagtgataa catccaccgg attggaaatg
     3241 accacaagca cgccatgaaa gccactttct tttagattgg ttcccacact ctggaccatg
     3301 gaggatgtga attttaattc agcaaatcta tccccggtgg gattatcttg ctgaagttta
     3361 atgtttccta aagtggaaat cacaacgtcg gcatcagcca aagccgccca atcattgata
     3421 acaatattcc catgcgcttc cagattcgcc atggcatcct gaaaatcaat ctgatctgct
     3481 ttcactttgg cctcgttcgc atcaataaac acataatcgt ctgcaacccc ttgcgcaatt
     3541 aagccgtggg caacggctgc gcctacgttg ccaaggccaa taatgccaat ttttcgtgcc
     3601 atatgtatat ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg
     3661 ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta
     3721 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
     3781 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
     3841 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
     3901 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     3961 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
     4021 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     4081 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     4141 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     4201 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     4261 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     4321 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     4381 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     4441 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     4501 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     4561 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     4621 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     4681 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     4741 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     4801 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     4861 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     4921 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     4981 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     5041 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     5101 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     5161 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
     5221 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     5281 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg
     5341 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg
     5401 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt
     5461 ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg
     5521 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     5581 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     5641 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     5701 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     5761 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     5821 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     5881 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     5941 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     6001 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     6061 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc
     6121 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     6181 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     6241 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cgg
//
