LOCUS       sequence-54827-e        6489 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 6489)
  TITLE     Kuwabara unpublished plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6489)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pPK719
FEATURES             Location/Qualifiers
     source          1..6489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..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     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     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     677..693
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(880..891)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(2910..2943)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     promoter        2947..2994
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(4176..4192)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4201..4234)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     primer_bind     complement(4301..4321)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4301..4319)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4340..4356)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4340..4356)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4353..4375)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4364..4380
                     /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(4388..4394,4395..4412,4413..4418))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4433..4454
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4571..4588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4742..5330)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4822..4841)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5501..6292,6293..6361))
                     /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     6124..6143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6362..6466)
                     /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 gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgataagat
      721 ctatgcttgc gctttgagcc aatttatcca agtccttgta aaaagtattc gaaaattgtt
      781 aacggataaa atgtttatta taatatcaaa aacaatttgt cagttgacca ctttttgata
      841 tatttgacag aaaccgggat gaattggctc aaagttaggg cgcccttcta ttacagggtt
      901 tctgataaca aaccggttat taactcccaa caagggatga tttcaattca tcatgctcaa
      961 attgacccaa attaagttac atgacaaatt catcgccctt ttcaactctt cttggctcat
     1021 catctgttat tctgttccta ttctgtctgc accctatacc cctttgcata ctctctcgtc
     1081 attcctttct tggatagtgt cttcttctcc ccagctctgc tacttctatg acttgcgccc
     1141 gctgcttttc cgctcgttcc tctcctctct gacgtcttct tctgcttctt ttctccttcc
     1201 tttttccctt ctcaatcttc ctctttttcc catttctgtc aatcattcga agaagaagaa
     1261 gaagaagacc ctcattcatt tcattttttt ttctgtcggt gtgtgctgct cggttaagag
     1321 tgagctctct attccacgtc ttcttctttt tcttcttcga ttcgaatcaa tcactccacc
     1381 aaaacgcatt cgtttttggg attcaccccg cggttcgcaa caggtttctt tttcaaatta
     1441 ttagcgttat aaatagaaaa atggtggagt ttcaataaaa atgataattt acaaagtgta
     1501 ttttgattac atgtactcaa aaaggttgaa atttttcata ccagttttcc ggaaatccat
     1561 ctgatatcat tatcgtattt ttctttttaa aaatgttttc aaaaaaaaac aaaatatagc
     1621 tggttatttg gcaccctcta attaccattt tcctgtcaca ccacactctt ttccttcttc
     1681 cacttctttc cgttttcagc cgcttccaac caaaccgata tgaaagccga gcaacaacaa
     1741 tcgattccac ccggctcggc gaccttcccg tcgcaggtga gactcagaaa actagagaaa
     1801 ccgctcaact aactcttgat atccgatttc attcttttct cttttctttt tgttgaactt
     1861 ttccatattt ccagatgcca cggccaccac caagcaccga acaaggaatc acaacggaat
     1921 cggagcttgc gaagaaagct caaatcactc cgaacgacgt tttagcactt ccgggaatca
     1981 ctcaaggtat cttgctccct ttggatttca gtttaaaaca taaatttagg attcttatgc
     2041 tccccatctg cgaacatcta taacatcgag ttcaccaagt tccaaatccg tgatctggac
     2101 actgagcaag tgcttttcga gatcgccaaa ccggagaacg atcaggagaa tgatgagtcg
     2161 ccacaggagt cggcaagata cgtgcgttat agatttgctc caaacttttt gaaactcaaa
     2221 acggtcggag caactgtgga attcaaagta ggagacatcc caatccatca tttccgaatg
     2281 atcgaacgtc acttcttcaa agatcgcctt ctgaagtgtt ttgactttga attcggattc
     2341 tgtattccga attcacgaaa caactgtgaa catatctatg agttccctca actctctcaa
     2401 caactcagtg agtcattatt ctaaaaagta caattcaaaa gactaatctc tttttcagtg
     2461 gacgacatga tcaacaatcc aaacgagact cgttctgaca gcttctattt cgtcgataac
     2521 aaactcgtca tgcacaacaa agccgactac tcatatgatg cataaatatt taatacaaaa
     2581 atgttctgga taattattct gtcgaataga aaaaaaactc caaatgtgat taaattccaa
     2641 taattcctgt ctagtttgtt ccttcccctt ccccttctca tgttcaatgc attcctaagc
     2701 ttttcagttc cccccttgtt tctatatttt ttcgctgtcc tgtcacactc gctaaaaaca
     2761 ctaatcacac ggaaatctgt tttcaataaa aactccaact ttaactcatt ttcaatttca
     2821 actgaaagat tttttcatta gagaatgtcc agcgaggagc tcgagtatca agcttatcga
     2881 taccgtcgac agtttaaaca gttcggtacg aagttcctat actatttgaa gaataggaac
     2941 ttccctgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
     3001 aggaactaaa cccaggaggc agatcatgag tctgaaagaa aaaacacaat ctctgtttgc
     3061 caacgcattt ggctaccctg ccactcacac cattcaggcg cctggccgcg tgaatttgat
     3121 tggtgaacac accgactaca acgacggttt cgttctgccc tgcgcgattg attatcaaac
     3181 cgtgatcagt tgtgcaccac gcgatgaccg taaagttcgc gtgatggcag ccgattatga
     3241 aaatcagctc gacgagtttt ccctcgatgc gcccattgtc gcacatgaaa actatcaatg
     3301 ggctaactac gttcgtggcg tggtgaaaca tctgcaactg cgtaacaaca gcttcggcgg
     3361 cgtggacatg gtgatcagcg gcaatgtgcc gcagggtgcc gggttaagtt cttccgcttc
     3421 actggaagtc gcggtcggaa ccgtattgca gcagctttat catctgccgc tggacggcgc
     3481 acaaatcgcg cttaacggtc aggaagcaga aaaccagttt gtaggctgta actgcgggat
     3541 catggatcag ctaatttccg cgctcggcaa gaaagatcat gccttgctga tcgattgccg
     3601 ctcactgggg accaaagcag tttccatgcc caaaggtgtg gctgtcgtca tcatcaacag
     3661 taacttcaaa cgtaccctgg ttggtagcga atacaacacc cgtcgtgaac agtgcgaaac
     3721 cggtgcgcgt ttcttccagc agccagccct gcgtgatgtc accattgaag agttcaacgc
     3781 tgttgcgcat gaactggacc cgatcgtgac aaaacgcgtg cgtcatatac tgactgaaaa
     3841 cgcccgcacc gttgaagctg ccagcgcgct ggagcaaggc gacctgaaac gtatgggcga
     3901 gttgatggcg gagtctcatg cctctatgcg cgatgatttc gaaatcaccg tgccgcaaat
     3961 tgacactctg gtagaaatcg tcaaagctgt gattggcgac aaaggtggcg tacgcatgac
     4021 cggcggcgga tttggcggct gtatcgtcgc gctgatcccg gaagagctgg tgcctgccgt
     4081 acagcaagct gtcgctgaac aatatgaagc aaaaacaggt attaaagaga ctttttacgt
     4141 ttgtaaacca tcacaaggag caggacagtg ctgaggatcc actagttcta gagcggccgc
     4201 gaagttccta tactatttga agaataggaa cttctaacta accatacata tttaaatttt
     4261 cgtcgacctc gagggggggc ccggtaccca gcttttgttc cctttagtga gggttaattg
     4321 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4381 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4441 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4501 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4561 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4621 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4681 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4741 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4801 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4861 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4921 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4981 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5041 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5101 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5161 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     5221 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5281 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5341 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5401 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5461 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5521 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5581 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5641 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5701 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5761 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5821 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5881 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5941 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6001 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6061 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6121 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6181 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6241 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6301 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6361 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6421 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6481 aagtgccac
//
