LOCUS       sequence-9739-ex        4678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4678)
  AUTHORS   Wang S, Zhao Y, Leiby M, Zhu J
  TITLE     A new positive/negative selection scheme for precise BAC 
            recombineering.
  JOURNAL   Mol Biotechnol. 2009 May;42(1):110-6. doi: 
            10.1007/s12033-009-9142-3. Epub 2009 Jan 22.
  PUBMED    19160076
REFERENCE   2  (bases 1 to 4678)
  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: 
            "10.1007/s12033-009-9142-3"; journalName: "Mol Biotechnol"; date: 
            "2009-05"; volume: "42"; issue: "1"; pages: "110-6"
COMMENT     Sequence Label: pSK+KanaRpsL
FEATURES             Location/Qualifiers
     source          1..4678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1446..2240
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1500..1519)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2110..2129
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2434..2450)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2434..2450)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(2487..2505)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2526..2542)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2526..2542)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2539..2561)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2550..2566
                     /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(2574..2580,2581..2598,2599..2604))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2619..2640
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2757..2774)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2928..3516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3008..3027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3687..4478,4479..4547))
                     /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     4310..4329
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4548..4652)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggaaattgta aacgttaata 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 gggcgcgtcg cgccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttcacgct gccgcaagca ctcagggcgc
      721 aagggctgct aaaggaagcg gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc
      781 cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag
      841 caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca
      901 agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta
      961 aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag atctaaccag
     1021 gagctattta atggcaacag ttaaccagct ggtacgcaaa ccacgtgctc gcaaagttgc
     1081 gaaaagcaac gtgcctgcgc tggaagcatg cccgcaaaaa cgtggcgtat gtactcgtgt
     1141 atatactacc actcctaaaa aaccgaactc cgcgctgcgt aaagtatgcc gtgttcgtct
     1201 gactaacggt ttcgaagtga cttcctacat cggtggtgaa ggtcacaacc tgcaggagca
     1261 ctccgtgatc ctgatccgtg gcggtcgtgt taaagacctc ccgggtgttc gttaccacac
     1321 cgtacgtggt gcgcttgact gctccggcgt taaagaccgt aagcaggctc gttccaagta
     1381 tggcgtgaag cgtcctaagg cttaatggta gatctgatca agagacagga tgacggtcgt
     1441 ttcgcatgct tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     1501 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     1561 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     1621 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     1681 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     1741 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     1801 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     1861 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     1921 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     1981 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     2041 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     2101 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     2161 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     2221 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     2281 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     2341 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     2401 cttcgcccac cccgggctcg atcccctcgg ggggatccac tagttctaga gcggccgcca
     2461 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattccgag cttggcgtaa
     2521 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     2581 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     2641 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2701 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     2761 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2821 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2881 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2941 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3001 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3061 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3121 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3181 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3241 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3301 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3361 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3421 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3481 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3541 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3601 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3661 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3721 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3781 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3841 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3901 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3961 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4021 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4081 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4141 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4201 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4261 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4321 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4381 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4441 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4501 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4561 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4621 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctg
//
