LOCUS Exported 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 http://www.snapgene.com 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 //