LOCUS       sequence-327824-        5422 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 5422)
  AUTHORS   Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH
  TITLE     Real-time observation of transcription initiation and elongation on 
            an endogenous yeast gene.
  JOURNAL   Science. 2011 Apr 22;332(6028):475-8.
  PUBMED    21512033
REFERENCE   2  (bases 1 to 5422)
  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: "Science."; 
            date: "2011-04-22"; volume: "332(6028)"; pages: "475-8"
COMMENT     Sequence Label: pCR4-24XPP7SL
FEATURES             Location/Qualifiers
     source          1..5422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1496..1515)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1497..1515)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1522..1539)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1522..1538)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1531..1553)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1676..1978
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1857..1876
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2327..3121
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2327..3121
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2381..2400)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2991..3010
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(3371..4162,4163..4231))
                     /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     3994..4013
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4355..4943
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4844..4863
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5097..5114
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5231..5252
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5267..5272,5273..5290,5291..5297)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5305..5321
                     /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)."
     primer_bind     5310..5332
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5329..5345
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5329..5345
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5366..5384
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ggatcctaag gtacctaatt gcctagaaag gagcagacga tatggcgtcg ctccctgcag
       61 gtcgactcta gaaaccagca gagcatatgg gctcgctggc tgcagtattc ccgggttcat
      121 tagatcctaa ggtacctaat tgcctagaaa ggagcagacg atatggcgtc gctccctgca
      181 ggtcgactct agaaaccagc agagcatatg ggctcgctgg ctgcagtatt cccgggttca
      241 ttagatccta aggtacctaa ttgcctagaa aggagcagac gatatggcgt cgctccctgc
      301 aggtcgactc tagaaaccag cagagcatat gggctcgctg gctgcagtat tcccgggttc
      361 attagatcct aaggtaccta attgcctaga aaggagcaga cgatatggcg tcgctccctg
      421 caggtcgact ctagaaacca gcagagcata tgggctcgct ggctgcagta ttcccgggtt
      481 cattagatcc taaggtacct aattgcctag aaaggagcag acgatatggc gtcgctccct
      541 gcaggtcgac tctagaaacc agcagagcat atgggctcgc tggctgcagt attcccgggt
      601 tcattagatc ctaaggtacc taattgccta gaaaggagca gacgatatgg cgtcgctccc
      661 tgcaggtcga ctctagaaac cagcagagca tatgggctcg ctggctgcag tattcccggg
      721 ttcattagat cctaaggtac ctaattgcct agaaaggagc agacgatatg gcgtcgctcc
      781 ctgcaggtcg actctagaaa ccagcagagc atatgggctc gctggctgca gtattcccgg
      841 gttcattaga tcctaaggta cctaattgcc tagaaaggag cagacgatat ggcgtcgctc
      901 cctgcaggtc gactctagaa accagcagag catatgggct cgctggctgc agtattcccg
      961 ggttcattag atcctaaggt acctaattgc ctagaaagga gcagacgata tggcgtcgct
     1021 ccctgcaggt cgactctaga aaccagcaga gcatatgggc tcgctggctg cagtattccc
     1081 gggttcatta gatcctaagg tacctaattg cctagaaagg agcagacgat atggcgtcgc
     1141 tccctgcagg tcgactctag aaaccagcag agcatatggg ctcgctggct gcagtattcc
     1201 cgggttcatt agatcctaag gtacctaatt gcctagaaag gagcagacga tatggcgtcg
     1261 ctccctgcag gtcgactcta gaaaccagca gagcatatgg gctcgctggc tgcagtattc
     1321 ccgggttcat tagatcctaa ggtacctaat tgcctagaaa ggagcagacg atatggcgtc
     1381 gctccctgca ggtcgactct agaaaccagc agagcatatg ggctcgctgg ctgcagtatt
     1441 cccgggttca ttagatctcg cgaagggcga attcgcggcc gctaaattca attcgcccta
     1501 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     1561 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     1621 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt
     1681 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga
     1741 tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc
     1801 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat
     1861 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct
     1921 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat
     1981 gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag
     2041 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga
     2101 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga
     2161 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa
     2221 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg
     2281 caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     2341 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     2401 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     2461 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     2521 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     2581 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     2641 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     2701 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     2761 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     2821 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     2881 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     2941 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3001 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     3061 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     3121 aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3181 ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3241 ttttctaaat acattcaaat atgtatccgc tcatgagatt atcaaaaagg atcttcacct
     3301 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3361 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3421 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3481 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3541 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3601 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3661 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3721 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3781 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3841 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3901 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3961 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4021 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4081 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4141 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4201 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4261 tttatcaggg ttattgtctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4321 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4381 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4441 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4501 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4561 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4621 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4681 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4741 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4801 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4861 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4921 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4981 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5041 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5101 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5161 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     5221 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     5281 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     5341 atgaccatga ttacgccaag ctcagaatta accctcacta aagggactag tcctgcaggt
     5401 ttaaacgaat tcgcccttcg cg
//
