LOCUS       sequence-473992-        6583 bp    DNA     circular SYN 28-MAY-2025
DEFINITION  Expression of His-tagged Thermococcus litoralis ADP hexokinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6583)
  TITLE     ADP-Hexokinase expression plasmid
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6583)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pMB-Tl-ADPHK
FEATURES             Location/Qualifiers
     source          1..6583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             29..58
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(81..99)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      95..142
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      179..634
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(266..285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     476..497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(749..1540,1541..1609))
                     /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     1372..1391
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1610..1702)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1783..2371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2272..2291
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2525..2542
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2557..2696
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2622..2641)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2782..2804
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2798..2989)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3561..4643)
                     /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     4605..4624
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4644..4721)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4927..4946
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5034..5053
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5034..5052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5053..5077
                     /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             5092..5114
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5130..5153
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5160..5174
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
ORIGIN
        1 agctccgggc tcctcctcaa tttccgctca tcaccaccat catcaccatc accaccacta
       61 attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
      121 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
      181 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
      241 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
      301 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
      361 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
      421 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
      481 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
      541 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
      601 gcgaatttta acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat
      661 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
      721 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
      781 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
      841 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
      901 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
      961 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     1021 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     1081 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     1141 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     1201 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     1261 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     1321 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     1381 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     1441 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     1501 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     1561 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     1621 ttcaatcatg attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     1681 tgtatttaga aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt
     1741 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1801 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1861 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1921 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     1981 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2041 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2101 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2161 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2221 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2281 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2341 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2401 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     2461 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     2521 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     2581 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     2641 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     2701 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2761 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2821 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     2881 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     2941 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3001 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3061 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     3121 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     3181 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     3241 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     3301 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     3361 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     3421 tcctcaacga caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga
     3481 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     3541 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3601 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     3661 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     3721 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     3781 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     3841 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     3901 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     3961 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     4021 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     4081 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     4141 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     4201 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     4261 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     4321 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     4381 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     4441 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     4501 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     4561 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     4621 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     4681 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     4741 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     4801 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     4861 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     4921 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     4981 ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga aattaatacg
     5041 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     5101 ctttaagaag gagatatacc atggcaagct ggagccaccc gcagttcgaa aagggtgcag
     5161 atgacgacga caaggtaccg gtgaaggaaa gccttaaaga taggataaga ttgtggaaaa
     5221 ggctgtatgt aaacgccttt gagaatgccc taaacgctat cccaaacgtt aagggtgttc
     5281 ttctggcata taacaccaac attgatgcca taaaatactt agacaaggac gatctagaaa
     5341 agagagtaac cgaaataggg aaagagaagg tttttgaaat tattgaaaat ccacctgaaa
     5401 agatctcctc cattgaagag cttcttggtg gaatattgag gagcataaaa cttggcaagg
     5461 ctatggagtg gtttgtagag agtgaggaag tgaggagata cttaagggaa tggggatggg
     5521 atgagctgag aataggaggg caggcaggga taatggcgaa ccttcttggg ggagtgtata
     5581 gaattccaac gattgttcat gttcctcaga atccaaagct tcaggcggag ttgtttgtag
     5641 atggtcctat ttatgtcccc gtctttgaag gaaataagtt gaagctagtt caccctaaag
     5701 atgccattgc agaggaagaa gagttaatcc actacatata tgaatttccc agaggttttc
     5761 aggtttttga tgttcaagca ccaagagaga atagattcat agccaatgcc gatgactaca
     5821 acgcgagggt ctacatgaga agggaattca gagaaggctt tgaggaaatt accagaaatg
     5881 ttgagctagc gataataagc gggctgcagg ttttaaagga atactatccc gacggaacaa
     5941 catacagaga tgttcttgat agagtcgaaa gtcatttgaa tatcctcaac cgctacaacg
     6001 tgaagagcca ttttgaattt gcatatactg caaacagaag ggttagggag gcacttgtag
     6061 agcttttacc gaagttcaca agcgtcggcc ttaatgaagt agaacttgcc tcaataatgg
     6121 agataatagg agacgaggag ctggcaaaag aagttctgga gggacatatc ttctcagtca
     6181 tagatgcgat gaatgtttta atggacgaga caggaattga gagaattcac ttccacacct
     6241 acggctacta cctcgcccta actcaataca gaggagaaga ggtaagagat gctctgctct
     6301 ttgcatccct cgctgcagct gccaaagcca tgaaaggaaa ccttgaaaga atagagcaga
     6361 ttagagatgc cctaagtgta ccaacaaatg aaagggcaat agtgcttgaa gaagaacttg
     6421 aaaaggagtt cacagaattt gagaacggcc ttattgatat ggtggacaga caacttgctt
     6481 ttgtgccaac aaagattgta gcatctccca agagcaccgt tggaattgga gataccattt
     6541 caagctctgc ttttgtcagt gaatttggca tgaggaaaag gaa
//
