LOCUS       pKSI-1                3032 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  cloning backbone.
ACCESSION   .
VERSION     .
KEYWORDS    pKSI-1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3032)
  AUTHORS   Yang J, Sun B, Huang H, Jiang Y, Diao L, Chen B, Xu C, Wang X, Liu 
            J, Jiang W, Yang S
  TITLE     High-Efficiency Scarless Genetic Modification in Escherichia coli by
            Using Lambda Red Recombination and I-SceI Cleavage.
  JOURNAL   Appl Environ Microbiol. 2014 Jul 1;80(13):3826-3834. Epub 2014 Apr 
            18.
  PUBMED    24747889
REFERENCE   2  (bases 1 to 3032)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            //gt12365.cn
FEATURES             Location/Qualifiers
     source          1..3032
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(471..887)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLPCYPYQASAGQLTLTKGNKSWVPGPPSRSTVSISL
                     ISNSCSPGDPLVLERPPPRWSSNSPYSESYYTIKALARDNRVMRAHWPSFYNVVTGKTL
                     ALPNLIALQHIPLSPAGVIAKRPAPIALPNSCAA"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     661..680
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        661..679
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    688..795
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     712..728
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     712..728
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(762..778)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(763..779)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(808..826)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(883..899)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(883..899)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(896..918)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    907..923
                     /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(931..961)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    976..997
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1114..1131)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1285..1873)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1365..1384)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2044..2904)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2667..2686
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2905..3009)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcattacc ctgttatccc tagccagcgc tttgatggtg
      661 taatacgact cactataggg cgaattggag ctccaccgcg gtggcggccg ctctagaact
      721 agtggatccc ccgggctgca ggaattcgat atcaagctta tcgataccgt cgacctcgag
      781 ggggggcccg gtacccagct tttgttccct ttagtgaggg ttaattggcc agcagaggcc
      841 tggtagggat aacagggtaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
      901 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
      961 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     1021 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1081 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     1141 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     1201 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1261 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     1321 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1381 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1441 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     1501 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1561 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1621 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1681 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     1741 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     1801 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     1861 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     1921 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     1981 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2041 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2101 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2161 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     2221 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2281 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2341 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     2401 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     2461 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     2521 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     2581 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     2641 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     2701 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     2761 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     2821 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     2881 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     2941 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3001 ggttccgcgc acatttcccc gaaaagtgcc ac
//