LOCUS       sequence-201132-        5334 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Cloning vector with temperature sensitive origin of replication..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5334)
  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 5334)
  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..5334
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(41..60)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             52..867
                     /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             52..867
                     /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     complement(125..144)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1719..1941
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             1989..2939
                     /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"
     primer_bind     4277..4294
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4411..4432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4447..4452,4453..4470,4471..4477)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4485..4501
                     /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     4490..4512
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4509..4525
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4509..4525
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4521..5096
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFAG
                     FPRQALNRGLPLGFRFSALRHLDPKKLD"
     primer_bind     4544..4564
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4546..4564
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    4577..4684
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     4601..4617
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4601..4617
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4651..4667)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4652..4668)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(4692..4711)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4693..4711)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4721..4738)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4721..4737)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4730..4752)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4879..5334
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4966..4985)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5176..5197
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
       61 attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta
      121 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
      181 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
      241 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
      301 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga
      361 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
      421 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
      481 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
      541 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
      601 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
      661 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
      721 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
      781 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
      841 catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag
      901 agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact tttgctgagt
      961 tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca aagcaaaagt
     1021 tcaaaatcac caactggtcc acctacaaca aagctctcat caaccgtggc tccctcactt
     1081 tctggctgga tgatggggcg attcagggca gtttgtctgc aagactctat gagaagcaga
     1141 taagcgataa gtttgctcaa catcttctcg ggcataagtc ggacaccatg accaaaatcc
     1201 cttaacgtga gttttcgttc cactgagcgt cagatccccg acagtaagac gggtaagcct
     1261 gttgatgata ccgctgcctt actgggtgca ttagccagtc tgaatgacct gtcacgggat
     1321 aatccgaagt ggtcagactg gaaaatcaga gggcaggaac tgctgaacag caaaaagtca
     1381 gatagcacca catagcagac ccgccataaa acgccctgag aagcccgtga cgggcttttc
     1441 ttgtattatg ggtagtttcc ttgcatgaat ccataaaagg cgcctgtagt gccatttacc
     1501 cccattcact gccagagccg tgagcgcagc gaactgaatg tcacgaaaaa gacagcgact
     1561 caggtgcctg atggtcggag acaaaaggaa tattcagcga tttgcccgag cttgcgaggg
     1621 tgctacttaa gcctttaggg ttttaaggtc tgttttgtag aggagcaaac agcgtttgcg
     1681 acatcctttt gtaatactgc ggaactgact aaagtagtga gttatacaca gggctgggat
     1741 ctattctttt tatctttttt tattctttct ttattctata aattataacc acttgaatat
     1801 aaacaaaaaa aacacacaaa ggtctagcgg aatttacaga gggtctagca gaatttacaa
     1861 gttttccagc aaaggtctag cagaatttac agatacccac aactcaaagg aaaaggacta
     1921 gtaattatca ttgactagcc catctcaatt ggtatagtga ttaaaatcac ctagaccaat
     1981 tgagatgtat gtctgaatta gttgttttca aagcaaatga actagcgatt agtcgctatg
     2041 acttaacgga gcatgaaacc aagctaattt tatgctgtgt ggcactactc aaccccacga
     2101 ttgaaaaccc tacaaggaaa gaacggacgg tatcgttcac ttataaccaa tacgttcaga
     2161 tgatgaacat cagtagggaa aatgcttatg gtgtattagc taaagcaacc agagagctga
     2221 tgacgagaac tgtggaaatc aggaatcctt tggttaaagg ctttgagatt ttccagtgga
     2281 caaactatgc caagttctca agcgaaaaat tagaattagt ttttagtgaa gagatattgc
     2341 cttatctttt ccagttaaaa aaattcataa aatataatct ggaacatgtt aagtcttttg
     2401 aaaacaaata ctctatgagg atttatgagt ggttattaaa agaactaaca caaaagaaaa
     2461 ctcacaaggc aaatatagag attagccttg atgaatttaa gttcatgtta atgcttgaaa
     2521 ataactacca tgagtttaaa aggcttaacc aatgggtttt gaaaccaata agtaaagatt
     2581 taaacactta cagcaatatg aaattggtgg ttgataagcg aggccgcccg actgatacgt
     2641 tgattttcca agttgaacta gatagacaaa tggatctcgt aaccgaactt gagaacaacc
     2701 agataaaaat gaatggtgac aaaataccaa caaccattac atcagattcc tacctacata
     2761 acggactaag aaaaacacta cacgatgctt taactgcaaa aattcagctc accagttttg
     2821 aggcaaaatt tttgagtgac atgcaaagta agtatgatct caatggttcg ttctcatggc
     2881 tcacgcaaaa acaacgaacc acactagaga acatactggc taaatacgga aggatctgag
     2941 gttcttatgg ctcttgtatc tatcagtgaa gcatcaagac taacaaacaa aagtagaaca
     3001 actgttcacc gttacatatc aaagggaaaa ctgtccatat gcacagatga aaacggtgta
     3061 aaaaagatag atacatcaga gcttttacga gtttttggtg catttaaagc tgttcaccat
     3121 gaacagatcg acaatgtaac agatgaacag catgtaacac ctaatagaac aggtgaaacc
     3181 agtaaaacaa agcaactaga acatgaaatt gaacacctga gacaacttgt tacagctcaa
     3241 cagtcacaca tagacagcct gaaacaggcg atgctgctta tcgaatcaaa gctgccgaca
     3301 acacgggagc cagtgacgcc tcccgtgggg aaaaaatcat ggcaattctg gaagaaatag
     3361 cgctttcagc cggcaaaccg gctgaagccg gatctgcgat tctgataaca aactagcaac
     3421 accagaacag cccgtttgcg ggcagcaaaa cccgtacttt tggacgttcc ggcggttttt
     3481 tgtggcgagt ggtgttcggg cggtgcgcgc aagatccatt atgttaaacg ggcgagttta
     3541 catctcaaaa ccgcccgctt aacaccatca gaaatcctca gcgcgatttt aagcaccaac
     3601 ccccccccgt aacacccaaa tccatactga aagtggcttt gttgaataaa tcgaactttt
     3661 gctgagttga aggatcagat cacgcatcct cccgacaaca cagaccattc cgtggcaaag
     3721 caaaagttca gaatcaccaa ctggtccacc tacaacaaag ctctcatcaa ccgtggctcc
     3781 ctcactttct ggctggatga tgaggcgatt caggcctggt atgagtcggc aacaccttca
     3841 tcacgaggaa ggccccagcg ctattctgat ctcgccatca ccaccgttct ggtgattaaa
     3901 cgcgtattcc ggctgaccct gcgggctgcg cagggtttta ttgattccat ttttgccctg
     3961 atgaacgttc cgttgcgctg cccggattac accagtgtca gtaagcgggc aaagtcggtt
     4021 aatgtcagtt tcaaaacgtc cacccggggt gaaatcgcac acctggtgat tgattccacc
     4081 gggctgaagg tctttggtga aggcgaatgg aaagtcagaa agcacggcaa agagcgccgt
     4141 cgtatctggc gaaagttgca tcttgctgtg gggatctcac atgttctttc ctgcgttatc
     4201 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4261 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     4321 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4381 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     4441 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     4501 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
     4561 aagggaacaa aagctggagc tccaccgcgg tggcggccgc tctagaacta gtggatcccc
     4621 cgggctgcag gaattcgata tcaagcttat cgataccgtc gacctcgagg gggggcccgg
     4681 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     4741 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4801 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4861 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4921 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4981 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5041 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5101 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5161 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5221 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5281 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca attt
//
