LOCUS       sequence-242393-        5946 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Azotobacter vinelandii flavodoxin II of Nitrogenase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5946)
  AUTHORS   Segal HM, Spatzal T, Hill MG, Udit AK, Rees DC
  TITLE     Electrochemical and structural characterization of Azotobacter 
            vinelandii flavodoxin II.
  JOURNAL   Protein Sci. 2017 Oct;26(10):1984-1993. doi: 10.1002/pro.3236. Epub 
            2017 Aug 30.
  PUBMED    28710816
REFERENCE   2  (bases 1 to 5946)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1002/pro.3236"; 
            journalName: "Protein Sci"; date: "2017-10"; volume: "26"; issue: 
            "10"; pages: "1984-1993"
COMMENT     Sequence Label: nifF in pET21b
FEATURES             Location/Qualifiers
     source          1..5946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     688..707
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(727..804)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1010..1029
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1113..1132
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1113..1131
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1132..1156
                     /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)."
     RBS             1171..1193
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1790..1807
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1860..1878)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1874..1921
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1958..2413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2045..2064)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2255..2276
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2439..2543
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2544..2612,2613..3404)
                     /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     complement(2762..2781)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3575..4163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4064..4083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4317..4334
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4349..4488
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4414..4433)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4574..4596
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4590..4781)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5590..5946,1..726))
                     /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"
ORIGIN
        1 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
       61 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
      121 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
      181 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
      241 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
      301 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
      361 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
      421 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
      481 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
      541 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
      601 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
      661 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
      721 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
      781 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
      841 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
      901 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
      961 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     1021 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     1081 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     1141 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
     1201 atggcgaaaa tcggcctgtt ctttggcagc aataccggca agacccgtaa ggtggcgaag
     1261 agcattaaaa aacgctttga cgacgaaacc atgagcgacg cgctgaacgt gaaccgtgtt
     1321 agcgcggagg atttcgcgca gtaccaattt ctgattctgg gcaccccgac cctgggcgag
     1381 ggtgaactgc cgggcctgag cagcgattgc gagaacgaaa gctgggagga gttcctgccg
     1441 aagattgaag gtctggactt cagcggcaaa accgtggcgc tgtttggtct gggcgaccag
     1501 gttggttacc cggagaacta tctggatgcg ctgggcgaac tgtacagctt ctttaaggac
     1561 cgtggtgcga aaatcgtggg cagctggagc accgatggtt atgagttcga aagcagcgag
     1621 gcggtggttg acggcaagtt tgttggcctg gcgctggacc tggataacca aagcggcaaa
     1681 accgatgagc gtgtggcggc gtggctggcg caaatcgcgc cggagttcgg tctgagcctg
     1741 taaggatccg aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca
     1801 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     1861 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1921 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     1981 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2041 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2101 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2161 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2221 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2281 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2341 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2401 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2461 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2521 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2581 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2641 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2701 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2761 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     2821 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2881 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2941 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3001 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3061 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     3121 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3181 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3241 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3301 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3361 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3421 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3481 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3541 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3601 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3661 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3721 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3781 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3841 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3901 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3961 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4021 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4081 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4141 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4201 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4261 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4321 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4381 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4441 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4501 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4561 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4621 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4681 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4741 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4801 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4861 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4921 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4981 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     5041 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5101 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     5161 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5221 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     5281 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     5341 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     5401 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     5461 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5521 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5581 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5641 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5701 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5761 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5821 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     5881 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5941 aaccag
//
