LOCUS       sequence-64912-e        4256 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 4256)
  AUTHORS   Monnat RJ Jr, Hackmann AF, Cantrell MA
  TITLE     Generation of highly site-specific DNA double-strand breaks in human
            cells by the homing endonucleases I-PpoI and I-CreI.
  JOURNAL   Biochem Biophys Res Commun. 1999 Feb 5;255(1):88-93.
  PUBMED    10082660
REFERENCE   2  (bases 1 to 4256)
  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: "Biochem 
            Biophys Res Commun."; date: "1999-02-5"; volume: "255(1)"; pages: 
            "88-93"
COMMENT     Sequence Label: pCNPpo6-H98A
FEATURES             Location/Qualifiers
     source          1..4256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        27..330
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        331..534
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     485..505
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     531..555
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          672..768
                     /label=SV40 intron
                     /note="modified SV40 intron with splice donor and acceptor 
                     sites"
     CDS             838..858
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1395..1529
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1419..1438)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1473..1492
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1640..1656)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1640..1656)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1653..1675)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1664..1680
                     /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(1688..1694,1695..1712,1713..1718))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1733..1754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1871..1888)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2042..2630)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2122..2141)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2801..3592,3593..3661))
                     /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     3424..3443
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3662..3766)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3834..3852
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3890..3912)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4012..4031
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4225..4247
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4239..4256
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4240..4256
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaattcgagc ttgcatgcct gcaggtcgtt acataactta cggtaaatgg cccgcctggc
       61 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      121 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      181 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      241 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      301 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      361 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      421 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      481 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
      541 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
      601 cgggaccgat ccagcctccg gactctagag gatccggtac tcgaggaact gaaaaaccag
      661 aaagttaact ggtaagttta gtctttttgt cttttatttc aggtcccgga tccggtggtg
      721 gtgcaaatca aagaactgct cctcagtgga tgttgccttt acttctaggc ctgtacggaa
      781 gtgttacttc tgctctaaaa gctgcggaat tgtacccgcg gccaattcac catggctcca
      841 aaaaaaaagc gcaaagtggc gctcaccaat gctcaaatct tggctgtgat tgacagctgg
      901 gaagaaacag tcggtcagtt tccagtgata acgcaccatg taccattagg tggcggtctg
      961 caaggaacgc tccattgtta cgagatcccc ctagcagctc cttatggggt tggctttgct
     1021 aagaatgggc ctacccgctg gcaatacaaa cggacaatca atcaagtcgt ccacagatgg
     1081 ggatcccaca cagtcccttt tctattagaa ccggataaca tcaacggcaa aacgtgcaca
     1141 gcaagcgcgc tatgtcataa tactcgatgc cacaatccct tgcacttgtg ctgggagtca
     1201 ctagacgaca acaaaggcag aaactggtgc ccgggtccca acgggggatg tgtccatgcg
     1261 gtggtttgtt taaggcaggg tccgttgtac ggcccggggg cgactgtggc aggtcctcaa
     1321 caaaggggca gtcactttgt ggtataaggt ctgaggttac tagatccggg taccgagctc
     1381 gaattggccg cggggatcca gacatgataa gatacattga tgagtttgga caaaccacaa
     1441 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     1501 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     1561 aggttcaggg ggaggtgtgg gaggtttttt cggatcctct agagtcgacc tgcaggcatg
     1621 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1681 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     1741 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     1801 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     1861 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     1921 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     1981 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2041 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2101 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2161 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2221 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2281 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2341 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2401 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2461 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     2521 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2581 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2641 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2701 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     2761 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     2821 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2881 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     2941 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3001 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3061 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3121 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3181 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3241 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3301 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3361 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3421 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3481 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3541 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3601 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3661 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     3721 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     3781 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     3841 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     3901 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     3961 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     4021 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     4081 gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     4141 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     4201 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagt
//
