LOCUS       sequence-465766-        4963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid designed to assay the site-selective methylation protection 
            approach using the non-switchable methylases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4963)
  AUTHORS   Flores-Fernandez CN, Lin D, Robins K, O'Callaghan CA
  TITLE     UniClo: scarless hierarchical DNA assembly without sequence 
            constraint.
  JOURNAL   Nucleic Acids Res. 2025 Jun 20;53(12):gkaf548. doi: 
            10.1093/nar/gkaf548.
  PUBMED    40548934
REFERENCE   2  (bases 1 to 4963)
  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: "Nucleic 
            Acids Res."; date: "2025-06-20"; volume: "53(12):gkaf548. doi"; 
            pages: " 10.1093/nar/gkaf548"
COMMENT     Sequence Label: POC1423
FEATURES             Location/Qualifiers
     source          1..4963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    97..118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(133..138,139..156,157..163)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    171..187
                     /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     176..198
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     195..211
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     195..211
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             207..530
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    224..280
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(281..298)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(281..297)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(290..312)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(506..525)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    669..693
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(849..1997)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(2333..2921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2413..2432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3092..3883,3884..3952))
                     /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     3715..3734
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3953..4057)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4144..4775
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4867..4891
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 cactctgtgg tgcagtacct ctcacgactt ggtctcatgc cgcagctggc acgacaggtt
       61 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      121 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      181 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca
      241 ggtcgactct agaggatccc cgggtaccga gctcgaattc actggccgtc gttttacaac
      301 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
      361 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
      421 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
      481 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
      541 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
      601 cttacagaca agctgtgact tgtcttctga gacctgaaga gcacgaagtg gtttaaacta
      661 tcagtgtttg acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat
      721 aatcggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc
      781 caaccacagg gttccccaga tcaggcgctg gctgctgaac ccccagccgg aactgacccc
      841 acaaggccct agcgtttgca atgcaccagg tcatcattga cccaggcgtg ttccaccagg
      901 ccgctgcctc gcaactcttc gcaggcttcg ccgacctgct cgcgccactt cttcacgcgg
      961 gtggaatccg atccgcacat gaggcggaag gtttccagct tgagcgggta cggctcccgg
     1021 tgcgagctga aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg gtacttctcc
     1081 catatgaatt tcgtgtagtg gtcgccagca aacagcacga cgatttcctc gtcgatcagg
     1141 acctggcaac gggacgtttt cttgccacgg tccaggacgc ggaagcggtg cagcagcgac
     1201 accgattcca ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc ctgtaggcgc
     1261 gacaggcatt cctcggcctt cgtgtaatac cggccattga tcgaccagcc caggtcctgg
     1321 caaagctcgt agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt cgcgtactcc
     1381 aacacctgct gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc ggtgtaggtg
     1441 atcttcacgt ccttgttgac gtggaaaatg accttgtttt gcagcgcctc gcgcgggatt
     1501 ttcttgttgc gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat cgctcgcatc
     1561 gtgtccggcc acggcgcaat atcgaacaag gaaagctgca tttccttgat ctgctgcttc
     1621 gtgtgtttca gcaacgcggc ctgcttggcc tcgctgacct gttttgccag gtcctcgccg
     1681 gcggtttttc gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat cgacttcgcc
     1741 aaacctgccg cctcctgttc aagacgacgc gaacgctcca cggcggccga tggcgcgggc
     1801 agggcagggg gagccagttg cacgctgtcg cgctcgatct tggccgtagc ttgctggacc
     1861 atcgagccga cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg gcttgcgatg
     1921 gtttcggcat cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta tgccttccgg
     1981 tcaaacgtcc gattcattca ccctccttgc gggattgccc cgactcacgc cggggcaatg
     2041 tgcccttatt cctgatttga cccgcctggt gccttggtgt ccagataatc caccttatcg
     2101 gcaatgaagt cggtcccgta gaccgtctgg ccgtccttct cgtacttggt attccgaatc
     2161 ttgccctgca cgaataccag cgaccccttg cccaaatact tgccgtgggc ctcggcctga
     2221 gagccaaaac acttgatgcg gaagaagtcg gtgcgctcct gcttgtcgcc ggcatcgttg
     2281 cgccacatct aggatctgcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2341 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2401 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2461 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2521 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2581 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2641 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2701 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2761 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2821 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2881 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2941 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3001 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3061 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3121 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3181 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagaaccac
     3241 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3301 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3361 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3421 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3481 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3541 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3601 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3661 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3721 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3781 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3841 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3901 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3961 tttttcaata ttattgaagc atttatcaag gttattgtct catgagcgga tacatatttg
     4021 aatgtattta gaaaaataaa caaatagggg ttccgcgcac gaattggcca gcgctgccat
     4081 ttttggggtg aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggaggcccgc
     4141 gttagcgggc cgggagggtt cgagaagggg gggcaccccc cttcggcgtg cgcggtcacg
     4201 cgcacagggc gcagccctgg ttaaaaacaa ggtttataaa tattggttta aaagcaggtt
     4261 aaaagacagg ttagcggtgg ccgaaaaacg ggcggaaacc cttgcaaatg ctggattttc
     4321 tgcctgtgga cagcccctca aatgtcaata ggtgcgcccc tcatctgtca gcactctgcc
     4381 cctcaagtgt caaggatcgc gcccctcatc tgtcagtagt cgcgcccctc aagtgtcaat
     4441 accgcagggc acttatcccc aggcttgtcc acatcatctg tgggaaactc gcgtaaaatc
     4501 aggcgttttc gccgatttgc gaggctggcc agctccacgt cgccggccga aatcgagcct
     4561 gcccctcatc tgtcaacgcc gcgccgggtg agtcggcccc tcaagtgtca acgtccgccc
     4621 ctcatctgtc agtgagggcc aagttttccg cgaggtatcc acaacgccgg cggccgcggt
     4681 gtctcgcaca cggcttcgac ggcgtttctg gcgcgtttgc agggccatag acggccgcca
     4741 gcccagcggc gagggcaacc agcccggtga gcgtcgcaaa ggagatcctg atctgactga
     4801 tgggctgcct gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct
     4861 ggctggtggc aggatatatt gtggtgtaaa caaattgacg cttagacaac ttaataacac
     4921 attgcggacg tttttaatgt actggggtgg atgcagtggg ccc
//
