LOCUS       sequence-199005-        3043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  an ampR plasmid built from pBluescript: the multiple cloning site 
            (mcs) flanked by inverted head-to-head oriented I-SceI sites. 
            Genbank accession number is KU144822..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3043)
  AUTHORS   Hoshijima K, Jurynec MJ, Grunwald DJ
  TITLE     Precise Editing of the Zebrafish Genome Made Simple and Efficient.
  JOURNAL   Dev Cell. 2016 Mar 21;36(6):654-67. doi: 
            10.1016/j.devcel.2016.02.015.
  PUBMED    27003937
REFERENCE   2  (bases 1 to 3043)
  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"; doi: 
            "10.1016/j.devcel.2016.02"; journalName: "Dev Cell"; date: 
            "2016-03-21- 21"; volume: "36"; issue: "6"; pages: "654-67"
COMMENT     Sequence Label: pKHR4
FEATURES             Location/Qualifiers
     source          1..3043
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..23
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4..22
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     104..120
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     104..120
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(154..170)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(155..171)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(236..254)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(275..291)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(275..291)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(288..310)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    299..315
                     /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(323..329,330..347,348..353))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    368..389
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(506..523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(677..1265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(757..776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1436..2227,2228..2296))
                     /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     2059..2078
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2297..2401)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2427..2882)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2564..2585)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2776..2795
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3009..3031
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3023..3040
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3024..3040
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttgtaatacg actcactata gggcgaattg gagctccacc gcggtggcgg ccaggtcctt
       61 tagtcgggta ttcgggtagg gataacaggg taatggcggc cgctctagaa ctagtggatc
      121 ccccgggctg caggaattcg atatcaagct tatcgatacc gtcgacctcg agccgccatt
      181 accctgttat ccctacccga atacccgact aaaggaccgg taccagcttt tgttcccttt
      241 agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      301 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
      361 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
      421 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
      481 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
      541 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
      601 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
      661 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
      721 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
      781 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
      841 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
      901 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
      961 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1021 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1081 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1141 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1201 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1261 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1321 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1381 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     1441 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     1501 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     1561 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     1621 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     1681 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     1741 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     1801 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     1861 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     1921 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     1981 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2041 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     2101 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2161 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2221 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2281 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2341 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2401 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     2461 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2521 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2581 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2641 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2701 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2761 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2821 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2881 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     2941 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     3001 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaa
//
