LOCUS       sequence-428726-        5570 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses his-tagged PNK in E. coli hosts.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5570)
  TITLE     Bacterial expression plasmids for production of recombinant PNK
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5570)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET-hisPNK
FEATURES             Location/Qualifiers
     source          1..5570
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(404..451)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     447..465
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1425..1436)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(1449..1478)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1492..1514
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(1545..1564)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1546..1564)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1648..1667)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             3125..3316
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3310..3332)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3418..3557
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3473..3492
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3572..3589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3743..4331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3823..3842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4502..5293,5294..5362))
                     /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     5125..5144
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5363..5467)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5535..5553
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata
      241 tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg
      301 ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac
      361 cacacccgtc ctgtggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
      421 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
      481 cagcttcctt tcgggctttg ttagcagccg gatccttaaa aatctcccga agcgacttgc
      541 cagcattcaa caccgatacg acgccacatt tcaactactt gagttcggtc atcaatagct
      601 aatttcacgt caaaatgcgg tgcaatgtgt ttccagaaaa tttcttcttt aactacatcg
      661 tctttacggg tatcgccttg ttcgcgctga cattgcataa ctaatggaac gccagcaatg
      721 tcctcaaccc atttacgggt catacgataa tatttcgttg ggtcttcttt agttccactt
      781 tcacgacctg aaacgactac gatttgataa cccataagag catacatctt agacagttca
      841 acaaccatag gattgataac atcggtatcg catttttcaa ggtcataagg accacgacca
      901 ttcattttag ctagtgtacc atcaacatca aaaataactg cttttggttt accaggagtc
      961 ccattatata ctggaagacc gagatactct cgcatgcttt tatacattga acgtaaaaca
     1021 tcaattggta ctgctttagt tccgcgtttt gagttacgtt taaccaattc agtccaagga
     1081 acatcaaaca ctttatgttc aactttccag ccgtattctt tggcaaaagt ttcccatgct
     1141 aggcgacgtt caggattcag gttagtatct gaaatgatta ctcccttaac agaatcgcca
     1201 ccgtacagaa tacttttagc tgtatcaaac tgcataccag ttacgatacc ttctttcttt
     1261 ttggtatact tgtactcatc gcgttcttca tgcgccataa tagattggcg atagtcatca
     1321 cgattgatat tataaaaccc gggattctta gcaataaatt cacgagccca agtactctta
     1381 ccagaaccag gacagccaat agtcaaaata atctttttca tatgacgacc ttcgatatgg
     1441 ccgctgctgt gatgatgatg atgatgatga tgatgatggc ccatatgtat atctccttct
     1501 taaagttaaa caaaattatt tctagaggga aaccgttgtg gtctccctat agtgagtcgt
     1561 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     1621 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     1681 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     1741 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     1801 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     1861 gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     1921 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     1981 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     2041 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     2101 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc
     2161 tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc
     2221 ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag
     2281 atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga
     2341 tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt
     2401 tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg
     2461 catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac
     2521 cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc
     2581 ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg
     2641 cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     2701 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     2761 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     2821 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     2881 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     2941 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     3001 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     3061 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     3121 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     3181 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     3241 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     3301 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     3361 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     3421 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     3481 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     3541 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3601 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3661 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3721 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3781 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3841 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3901 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3961 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4021 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4081 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4141 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4201 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4261 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4321 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4381 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4441 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4501 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4561 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4621 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4681 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4741 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4801 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4861 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4921 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4981 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5041 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5101 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5161 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5221 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5281 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5341 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5401 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5461 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     5521 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa
//
