LOCUS       sequence-156252-        7383 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7383)
  AUTHORS   Narayana N, Cox S, Shaltiel S, Taylor SS, Xuong N
  TITLE     Crystal structure of a polyhistidine-tagged recombinant catalytic 
            subunit of cAMP-dependent protein kinase complexed with the peptide 
            inhibitor PKI(5-24) and adenosine.
  JOURNAL   Biochemistry. 1997 Apr 15. 36(15):4438-48.
  PUBMED    9109651
REFERENCE   2  (bases 1 to 7383)
  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"; journalName: 
            "Biochemistry. 1997 Apr 15. 36(15):4438-48."
COMMENT     Sequence Label: pET15b PKA Cat
FEATURES             Location/Qualifiers
     source          1..7383
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(2014..2031)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2041..2058)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2077..2099
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2114..2138
                     /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(2138..2157)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2139..2157)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2241..2260)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2466..2543
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2544..3626
                     /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(2563..2582)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4938..5129
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5123..5145)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5231..5370
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5286..5305
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5385..5402)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5556..6144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5636..5655)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6315..7106,7107..7175))
                     /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     6938..6957
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7176..7280)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7348..7366
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gcttgcatgc ctgcaggtcg actctagagg
       61 atccccgggt accgagctcc tcctttttgg gagaagttac gagagggata aacacaggga
      121 taaatggggt aatttcccca gcagctcccc acctttctat tgagagaaaa agcacaatca
      181 gacttggggt tgcagtgagt ggggacgcac ggcaagaggt gatggacagc tgacagctaa
      241 gctagaagcg aggcgcccag gatgggggag gcttcttaag ggggctgatg cacaggacaa
      301 ttggcatgag accactggaa aggggggccg ggaggctagc agctcagggc gagattccaa
      361 gcccagttcc ttccttgaca gggtcagttg gctacccagg tctcatcaga atggatttag
      421 ggagtgagga gggtgtttgg ggagggtggg acaatttgga gggtgggagg cactggtgga
      481 acttaattta aatgaaaaaa aaaacccact ggcaagcagc cctctgtaca ggtcataaaa
      541 tctccctttc caggtagggc tgcatggaag ggaaggttgg cgttactgtt cgttcatcct
      601 tagtatccag tgctagggtc gggaaggagg ctgaatttgg ggccctcttt ccgtaccctg
      661 cccctggata caccagcaag attgggtatg gctggagcta agaaattcat tgaaaacagg
      721 cagggaaggg tggagaagaa gggagagggg gagcagctga tgttgcccct tccccagtat
      781 ctggagcgct ttcctgctct ccttaaccct gggggtgtgg agtggatgtt aaattagatg
      841 catggaactc tagggccctc tggctgttcg atccaaccct cccaccccca accggaaaaa
      901 aaatagaaag aaaaagaaaa aatgaaagag aacccaaggg gcacaagcac acccctaaaa
      961 ctcagtaaac tccttgccac acttctcatt gatggagacc cggatctctt cctcctcata
     1021 gtcgtcaaag ttactcgtgt ccccagggcc tttaaacttt ggtatgaagg gagcttccac
     1081 ctttctctga tagatggcaa tccagtcagt cgtggcaaac cacttgtggt tcttgatgtc
     1141 attgaccccg ttcttgaggt tcccaaagcg cttggtgaga tccacttgca gaaggttccg
     1201 cagcaggtcc ttcaagtcag agctgaagtg ggatgggaac cgcaccttcc cagagacgat
     1261 tttctcatag atctggatag gctggtcagc gaagaagggt gggtaaccag cagccatctc
     1321 gtagatgagg actccgagag cccaccagtc cacagccttg ttgtagcctt tgctcaggat
     1381 aatctcgggg gccaagtact caggggtccc acacaaggtc caagtacggc ctttcacacg
     1441 cttggcaaaa ccgaagtctg tcacctgaat atagccctgc tggtcgatga gaagattctc
     1501 gggcttcagg tcccggtaga tgaggtccag ggagtgcaga tactcaaagg tcaggacgat
     1561 ctgcgccgcg tagaaacggg catggggctc gctgaacctt ccaatccgcc gtaggtggga
     1621 gaacatctcg ccaccagcta catactccat gaccatgtac aggtttgagt tgtccttgaa
     1681 ggagaattca agtttgacca ggaacgggaa gttgacggcc tgcaggatgc gcttctcatt
     1741 cagagtgtgc tcgatctgct ttagcttcac caccttctgc ttgtctaaga tcttcatggc
     1801 gtagtggttc ccactctcct tgtgcttcac cagcatcact cgcccaaagg agccggtgcc
     1861 aagggtcttg attctatcaa actgatccaa ctgggctgta ttctgagaag gggtctccca
     1921 ttttttcagg aaatcttcct tggctttggc taggaactct ttcacgctct cctgctcgct
     1981 gcccttcttg gcggcggcgg cgttgcccat atggctgccg cgcggcacca ggccgctgct
     2041 gtgatgatga tgatgatggc tgctgcccat ggtatatctc cttcttaaag ttaaacaaaa
     2101 ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt
     2161 tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
     2221 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
     2281 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg
     2341 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg
     2401 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga
     2461 tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     2521 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     2581 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     2641 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     2701 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     2761 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     2821 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     2881 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     2941 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     3001 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     3061 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     3121 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     3181 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     3241 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     3301 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     3361 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     3421 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     3481 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     3541 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     3601 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     3661 taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     3721 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc
     3781 gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc
     3841 gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc
     3901 ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg
     3961 gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc
     4021 cccattatga ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg
     4081 tccaggcagg tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc
     4141 agcctaactt cgatcattgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc
     4201 acatggaacg ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg
     4261 ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg
     4321 ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg
     4381 cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc
     4441 ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt
     4501 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
     4561 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
     4621 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
     4681 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
     4741 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
     4801 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
     4861 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
     4921 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
     4981 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
     5041 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
     5101 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     5161 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     5221 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     5281 ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac
     5341 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     5401 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5461 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5521 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5581 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5641 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5701 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5761 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5821 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5881 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5941 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     6001 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6061 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6121 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6181 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6241 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6301 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6361 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6421 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6481 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6541 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6601 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt
     6661 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6721 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6781 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6841 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6901 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     6961 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7021 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7081 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7141 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7201 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7261 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     7321 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa
     7381 gaa
//
