LOCUS       sequence-462636-        6141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Klebsiella pneumoniae carbapenemase-2 (KPC-2) under T7 
            with His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6141)
  AUTHORS   Beckham J
  TITLE     Josh Beckham Lab plasmids 2024
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6141)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: KPC2-pNic-Bsa4
FEATURES             Location/Qualifiers
     source          1..6141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1006..1023
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1076..1094)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1090..1137
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1174..1629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1261..1280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1471..1492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1721..2536)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1721..2536)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2444..2463
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2528..2547
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2658..3246
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3147..3166
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3400..3417
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3432..3571
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3497..3516)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3657..3679
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3673..3864)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4673..5755)
                     /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     5717..5736
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5756..5833)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6039..6058
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatggcg gaaccattcg ctaaactcga
      181 acaggacttt ggcggctcca tcggtgtgta cgcgatggat accggctcag gcgcaactgt
      241 aagttaccgc gctgaggagc gcttcccact gtgcagctca ttcaagggct ttcttgctgc
      301 cgctgtgctg gctcgcagcc agcagcaggc cggcttgctg gacacaccca tccgttacgg
      361 caaaaatgcg ctggttccgt ggtcacccat ctcggaaaaa tatctgacaa caggcatgac
      421 ggtggcggag ctgtccgcgg ccgccgtgca atacagtgat aacgccgccg ccaatttgtt
      481 gctgaaggag ttgggcggcc cggccgggct gacggccttc atgcgctcta tcggcgatac
      541 cacgttccgt ctggaccgct gggagctgga gctgaactcc gccatcccag gcgatgcgcg
      601 cgatacctca tcgccgcgcg ccgtgacgga aagcttacaa aaactgacac tgggctctgc
      661 actggctgcg ccgcagcggc agcagtttgt tgattggcta aagggaaaca cgaccggcaa
      721 ccaccgcatc cgcgcggcgg tgccggcaga ctgggcagtc ggagacaaaa ccggaacctg
      781 cggagtgtat ggcacggcaa atgactatgc cgtcgtctgg cccactgggc gcgcacctat
      841 tgtgttggcc gtctacaccc gggcgcctaa caaggatgac aagcacagcg aggccgtcat
      901 cgccgctgcg gctagactcg cgctcgaggg attgggcgtc taacagtaaa ggtggatacg
      961 gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac
     1021 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1081 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1141 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1201 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1261 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1321 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1381 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1441 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1501 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1561 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1621 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1681 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1741 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1801 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1861 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1921 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1981 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     2041 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     2101 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     2161 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     2221 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2281 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2341 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2401 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2461 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2521 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2581 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2641 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2701 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2761 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2821 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2881 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2941 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3001 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3061 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3121 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3181 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3241 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3301 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3361 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3421 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3481 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3541 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3601 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3661 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3721 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3781 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     3841 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     3901 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     3961 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4021 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4081 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4141 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4201 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4261 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4321 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4381 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4441 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4501 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4561 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4621 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4681 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4741 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4801 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4861 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4921 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     4981 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5041 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5101 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5161 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5221 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5281 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5341 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5401 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5461 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5521 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5581 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5641 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5701 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5761 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5821 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5881 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5941 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6001 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6061 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     6121 gagatctcga tcccgcgaaa t
//
