LOCUS       sequence-458378-        6526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human PTK2b Kinase domain in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6526)
  TITLE     Mesecar lab plasmids 2024
REFERENCE   2  (bases 1 to 6526)
  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"
COMMENT     Sequence Label: pET11a_PTK2b_Kinase
FEATURES             Location/Qualifiers
     source          1..6526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1265..2347)
                     /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     2309..2328
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2348..2425)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2631..2650
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2734..2753
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2734..2752
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2753..2777
                     /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             2792..2814
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2825..2842
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2843..2863
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3757..3775)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3771..3818
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3993..3998,3999..4015,4016..4021))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4048..4066)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4134..4238
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4239..4307,4308..5099)
                     /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     complement(4457..4476)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5270..5858
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5759..5778
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6012..6029
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6044..6186
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6112..6131)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6272..6294
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6288..6479)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
       61 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
      121 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
      181 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
      241 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
      301 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
      361 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
      421 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
      481 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg
      541 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
      601 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
      661 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
      721 gatcagcggt ccagtgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
      781 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
      841 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
      901 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
      961 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     1021 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     1081 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     1141 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     1201 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     1261 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1321 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     1381 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     1441 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     1501 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     1561 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     1621 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     1681 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     1741 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     1801 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     1861 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     1921 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     1981 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     2041 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     2101 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     2161 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     2221 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     2281 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     2341 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     2401 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     2461 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     2521 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     2581 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     2641 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     2701 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     2761 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca
     2821 tatgcaccac caccaccacc atgaaaacct gtacttccag ggtagcatgg gcggcccgca
     2881 gtacggtatc gcgcgtgagg acgttgttct gaaccgtatc ctgggtgaag gcttcttcgg
     2941 tgaagtttac gaaggcgttt acaccaacca caaaggtgaa aaaatcaacg ttgctgttaa
     3001 aacctgcaaa aaagactgca ccctggacaa caaagaaaaa ttcatgtctg aagctgttat
     3061 catgaaaaac ctggatcacc cgcacatcgt taaactgatc ggtatcatcg aagaagaacc
     3121 gacttggatc atcatggaac tgtacccgta cggtgaactg ggccactacc tggaacgtaa
     3181 caaaaactct ctgaaagttc tgaccctggt tctgtactct ctgcagatct gcaaagcgat
     3241 ggcgtacctg gaatctatca actgcgttca ccgtgacatc gctgttcgta acatcctggt
     3301 tgcgtccccg gaatgcgtta aactgggtga cttcggcctg tctcgttaca tcgaagatga
     3361 agattactac aaagcttccg ttacccgtct gccgatcaaa tggatgagcc cggaatctat
     3421 taacttccgc cgtttcacca ccgcgtctga cgtttggatg ttcgcggttt gcatgtggga
     3481 aatcctgtct ttcggtaaac agccgttctt ctggctggaa aacaaagatg tgatcggcgt
     3541 tctggaaaaa ggtgaccgtc tgccgaaacc ggatctgtgc ccgccggttc tgtacaccct
     3601 gatgacccgt tgctgggact acgatccgtc tgaccgtccg cgtttcaccg aactggtttg
     3661 ctctctgtct gacgtttacc agatggaaaa agacatcgcg atggaaggat ccggctgcta
     3721 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     3781 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     3841 gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag
     3901 ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact
     3961 gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca
     4021 aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4081 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4141 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4201 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4261 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4321 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4381 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4441 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
     4501 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4561 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4621 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4681 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4741 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     4801 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4861 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4921 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4981 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5041 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5101 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5161 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5221 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5281 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5341 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5401 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5461 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5521 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5581 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5641 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5701 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5761 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5821 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5881 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5941 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     6001 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     6061 tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg
     6121 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     6181 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     6241 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     6301 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     6361 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     6421 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     6481 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgatac
//
