LOCUS       sequence-262664-        5726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector with temperature sensitive origin of replication..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5726)
  AUTHORS   Phillips GJ
  TITLE     New cloning vectors with temperature-sensitive replication.
  JOURNAL   Plasmid. 1999 Jan;41(1):78-81.
  PUBMED    9887310
REFERENCE   2  (bases 1 to 5726)
  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: "Plasmid."; 
            date: "1999-01"; volume: "41(1)"; pages: "78-81"
COMMENT     Sequence Label: pTSK29
FEATURES             Location/Qualifiers
     source          1..5726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(979..1929)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      1977..2199
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(3051..3866)
                     /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(3051..3866)
                     /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     3774..3793
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3858..3877
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      complement(4310..4765)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4447..4468)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4548..5123)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFAG
                     FPRQALNRGLPLGFRFSALRHLDPKKLD"
     primer_bind     4659..4678
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4892..4914
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4906..4923
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4907..4923
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4933..4952
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4933..4951
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    4960..5067
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     4976..4992
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4977..4993
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5027..5043)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5027..5043)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5080..5100)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5080..5098)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5119..5135)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5119..5135)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5132..5154)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5143..5159
                     /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)."
     promoter        complement(join(5167..5173,5174..5191,5192..5197))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5212..5233
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5350..5367)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtcagccgga atacgcgttt aatcaccaga acggtggtga tggcgagatc agaatagcgc
       61 tggggccttc ctcgtgatga aggtgttgcc gactcatacc aggcctgaat cgcctcatca
      121 tccagccaga aagtgaggga gccacggttg atgagagctt tgttgtaggt ggaccagttg
      181 gtgattctga acttttgctt tgccacggaa tggtctgtgt tgtcgggagg atgcgtgatc
      241 tgatccttca actcagcaaa agttcgattt attcaacaaa gccactttca gtatggattt
      301 gggtgttacg ggggggggtt ggtgcttaaa atcgcgctga ggatttctga tggtgttaag
      361 cgggcggttt tgagatgtaa actcgcccgt ttaacataat ggatcttgcg cgcaccgccc
      421 gaacaccact cgccacaaaa aaccgccgga acgtccaaaa gtacgggttt tgctgcccgc
      481 aaacgggctg ttctggtgtt gctagtttgt tatcagaatc gcagatccgg cttcagccgg
      541 tttgccggct gaaagcgcta tttcttccag aattgccatg attttttccc cacgggaggc
      601 gtcactggct cccgtgttgt cggcagcttt gattcgataa gcagcatcgc ctgtttcagg
      661 ctgtctatgt gtgactgttg agctgtaaca agttgtctca ggtgttcaat ttcatgttct
      721 agttgctttg ttttactggt ttcacctgtt ctattaggtg ttacatgctg ttcatctgtt
      781 acattgtcga tctgttcatg gtgaacagct ttaaatgcac caaaaactcg taaaagctct
      841 gatgtatcta tcttttttac accgttttca tctgtgcata tggacagttt tccctttgat
      901 atgtaacggt gaacagttgt tctacttttg tttgttagtc ttgatgcttc actgatagat
      961 acaagagcca taagaacctc agatccttcc gtatttagcc agtatgttct ctagtgtggt
     1021 tcgttgtttt tgcgtgagcc atgagaacga accattgaga tcatacttac tttgcatgtc
     1081 actcaaaaat tttgcctcaa aactggtgag ctgaattttt gcagttaaag catcgtgtag
     1141 tgtttttctt agtccgttat gtaggtagga atctgatgta atggttgttg gtattttgtc
     1201 accattcatt tttatctggt tgttctcaag ttcggttacg agatccattt gtctatctag
     1261 ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc ttatcaacca ccaatttcat
     1321 attgctgtaa gtgtttaaat ctttacttat tggtttcaaa acccattggt taagcctttt
     1381 aaactcatgg tagttatttt caagcattaa catgaactta aattcatcaa ggctaatctc
     1441 tatatttgcc ttgtgagttt tcttttgtgt tagttctttt aataaccact cataaatcct
     1501 catagagtat ttgttttcaa aagacttaac atgttccaga ttatatttta tgaatttttt
     1561 taactggaaa agataaggca atatctcttc actaaaaact aattctaatt tttcgcttga
     1621 gaacttggca tagtttgtcc actggaaaat ctcaaagcct ttaaccaaag gattcctgat
     1681 ttccacagtt ctcgtcatca gctctctggt tgctttagct aatacaccat aagcattttc
     1741 cctactgatg ttcatcatct gaacgtattg gttataagtg aacgataccg tccgttcttt
     1801 ccttgtaggg ttttcaatcg tggggttgag tagtgccaca cagcataaaa ttagcttggt
     1861 ttcatgctcc gttaagtcat agcgactaat cgctagttca tttgctttga aaacaactaa
     1921 ttcagacata catctcaatt ggtctaggtg attttaatca ctataccaat tgagatgggc
     1981 tagtcaatga taattactag tccttttcct ttgagttgtg ggtatctgta aattctgcta
     2041 gacctttgct ggaaaacttg taaattctgc tagaccctct gtaaattccg ctagaccttt
     2101 gtgtgttttt tttgtttata ttcaagtggt tataatttat agaataaaga aagaataaaa
     2161 aaagataaaa agaatagatc ccagccctgt gtataactca ctactttagt cagttccgca
     2221 gtattacaaa aggatgtcgc aaacgctgtt tgctcctcta caaaacagac cttaaaaccc
     2281 taaaggctta agtagcaccc tcgcaagctc gggcaaatcg ctgaatattc cttttgtctc
     2341 cgaccatcag gcacctgagt cgctgtcttt ttcgtgacat tcagttcgct gcgctcacgg
     2401 ctctggcagt gaatgggggt aaatggcact acaggcgcct tttatggatt catgcaagga
     2461 aactacccat aatacaagaa aagcccgtca cgggcttctc agggcgtttt atggcgggtc
     2521 tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct gattttccag
     2581 tctgaccact tcggattatc ccgtgacagg tcattcagac tggctaatgc acccagtaag
     2641 gcagcggtat catcaacagg cttacccgtc ttactgtcgg ggatctgacg ctcagtggaa
     2701 cgaaaactca cgttaaggga ttttggtcat ggtgtccgac ttatgcccga gaagatgttg
     2761 agcaaactta tcgcttatct gcttctcata gagtcttgca gacaaactgc cctgaatcgc
     2821 cccatcatcc agccagaaag tgagggagcc acggttgatg agagctttgt tgtaggtgga
     2881 ccagttggtg attttgaact tttgctttgc cacggaacgg tctgcgttgt cgggaagatg
     2941 cgtgatctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc gccgtcccgt
     3001 caagtcagcg taatgctctg ccagtgttac aaccaattaa ccaattctga ttagaaaaac
     3061 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     3121 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     3181 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     3241 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     3301 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     3361 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     3421 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     3481 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     3541 acctggaatg ctgtttttcc ggggatcgca gtggtgagta accatgcatc atcaggagta
     3601 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     3661 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     3721 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     3781 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa
     3841 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     3901 agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga
     3961 gacaacgtgg ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc agatcacgca
     4021 tcttcccgac aacgcagacc gttccgtggc aaagcaaaag ttcaaaatca ccaactggtc
     4081 cacctacaac aaagctctca tcaaccgtgg ctccctcact ttctggctgg atgatggggc
     4141 gattcagggc agtttgtctg caagactcta tgagaagcag ataagcgata agtttgctca
     4201 acatcttctc gggcataagt cggacaccat gagcggatac atatttgaat gtatttagaa
     4261 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     4321 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     4381 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     4441 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     4501 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     4561 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     4621 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     4681 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     4741 cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa
     4801 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     4861 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     4921 agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag
     4981 gtcgacggta tcgataagct tgatatcgaa ttcctgcagc ccgggggatc cactagttct
     5041 agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc
     5101 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5161 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5221 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5281 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5341 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5401 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5461 catgtgagat ccccacagca agatgcaact ttcgccagat acgacggcgc tctttgccgt
     5521 gctttctgac tttccattcg ccttcaccaa agaccttcag cccggtggaa tcaatcacca
     5581 ggtgtgcgat ttcaccccgg gtggacgttt tgaaactgac attaaccgac tttgcccgct
     5641 tactgacact ggtgtaatcc gggcagcgca acggaacgtt catcagggca aaaatggaat
     5701 caataaaacc ctgcgcagcc cgcagg
//
