LOCUS       sequence-465737-        5029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Assembly vector designed to be methylated by the M.Osp807II 
            BsaI-associated switch methylase and for the assembly of inserts 1, 
            2, 3 and 4 from POC1426, POC1427, POC1428 and POC1429; respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5029)
  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 5029)
  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: POC1430
FEATURES             Location/Qualifiers
     source          1..5029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    124..145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(160..165,166..183,184..190)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    198..214
                     /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     203..225
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     222..238
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     222..238
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             234..557
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    251..307
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(308..325)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(308..324)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(317..339)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(533..552)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    735..759
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(915..2063)
                     /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(2399..2987)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2479..2498)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3158..3949,3950..4018))
                     /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     3781..3800
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4019..4123)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4210..4841
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4933..4957
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 cactctgtgg tgcagtacct ctcacgactt ggtctcacgt ttgagaccgg cctagcaggt
       61 gtgagtttgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      121 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      181 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      241 tgattacgcc aagcttgcat gcctgcaggt cgactctaga ggatccccgg gtaccgagct
      301 cgaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
      361 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
      421 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt
      481 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
      541 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
      601 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgacaagt accacctgga
      661 acgccaaagg tctcacaggt gagaccaagt cgtgagaggt actgcaccac gaagtggttt
      721 aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat
      781 tagaataatc ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt
      841 gcatgccaac cacagggttc cccagatcag gcgctggctg ctgaaccccc agccggaact
      901 gaccccacaa ggccctagcg tttgcaatgc accaggtcat cattgaccca ggcgtgttcc
      961 accaggccgc tgcctcgcaa ctcttcgcag gcttcgccga cctgctcgcg ccacttcttc
     1021 acgcgggtgg aatccgatcc gcacatgagg cggaaggttt ccagcttgag cgggtacggc
     1081 tcccggtgcg agctgaaata gtcgaacatc cgtcgggccg tcggcgacag cttgcggtac
     1141 ttctcccata tgaatttcgt gtagtggtcg ccagcaaaca gcacgacgat ttcctcgtcg
     1201 atcaggacct ggcaacggga cgttttcttg ccacggtcca ggacgcggaa gcggtgcagc
     1261 agcgacaccg attccaggtg cccaacgcgg tcggacgtga agcccatcgc cgtcgcctgt
     1321 aggcgcgaca ggcattcctc ggccttcgtg taataccggc cattgatcga ccagcccagg
     1381 tcctggcaaa gctcgtagaa cgtgaaggtg atcggctcgc cgataggggt gcgcttcgcg
     1441 tactccaaca cctgctgcca caccagttcg tcatcgtcgg cccgcagctc gacgccggtg
     1501 taggtgatct tcacgtcctt gttgacgtgg aaaatgacct tgttttgcag cgcctcgcgc
     1561 gggattttct tgttgcgcgt ggtgaacagg gcagagcggg ccgtgtcgtt tggcatcgct
     1621 cgcatcgtgt ccggccacgg cgcaatatcg aacaaggaaa gctgcatttc cttgatctgc
     1681 tgcttcgtgt gtttcagcaa cgcggcctgc ttggcctcgc tgacctgttt tgccaggtcc
     1741 tcgccggcgg tttttcgctt cttggtcgtc atagttcctc gcgtgtcgat ggtcatcgac
     1801 ttcgccaaac ctgccgcctc ctgttcaaga cgacgcgaac gctccacggc ggccgatggc
     1861 gcgggcaggg cagggggagc cagttgcacg ctgtcgcgct cgatcttggc cgtagcttgc
     1921 tggaccatcg agccgacgga ctggaaggtt tcgcggggcg cacgcatgac ggtgcggctt
     1981 gcgatggttt cggcatcctc ggcggaaaac cccgcgtcga tcagttcttg cctgtatgcc
     2041 ttccggtcaa acgtccgatt cattcaccct ccttgcggga ttgccccgac tcacgccggg
     2101 gcaatgtgcc cttattcctg atttgacccg cctggtgcct tggtgtccag ataatccacc
     2161 ttatcggcaa tgaagtcggt cccgtagacc gtctggccgt ccttctcgta cttggtattc
     2221 cgaatcttgc cctgcacgaa taccagcgac cccttgccca aatacttgcc gtgggcctcg
     2281 gcctgagagc caaaacactt gatgcggaag aagtcggtgc gctcctgctt gtcgccggca
     2341 tcgttgcgcc acatctagga tctgccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2401 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2461 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2521 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2581 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2641 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2701 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2761 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2821 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2881 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2941 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3001 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3061 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3121 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3181 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3241 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3301 aaccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3361 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3421 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3481 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3541 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3601 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3661 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3721 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3781 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3841 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3901 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3961 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4021 tcttcctttt tcaatattat tgaagcattt atcaaggtta ttgtctcatg agcggataca
     4081 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacgaat tggccagcgc
     4141 tgccattttt ggggtgaggc cgttcgcggc cgaggggcgc agcccctggg gggatgggag
     4201 gcccgcgtta gcgggccggg agggttcgag aagggggggc accccccttc ggcgtgcgcg
     4261 gtcacgcgca cagggcgcag ccctggttaa aaacaaggtt tataaatatt ggtttaaaag
     4321 caggttaaaa gacaggttag cggtggccga aaaacgggcg gaaacccttg caaatgctgg
     4381 attttctgcc tgtggacagc ccctcaaatg tcaataggtg cgcccctcat ctgtcagcac
     4441 tctgcccctc aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg cccctcaagt
     4501 gtcaataccg cagggcactt atccccaggc ttgtccacat catctgtggg aaactcgcgt
     4561 aaaatcaggc gttttcgccg atttgcgagg ctggccagct ccacgtcgcc ggccgaaatc
     4621 gagcctgccc ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa gtgtcaacgt
     4681 ccgcccctca tctgtcagtg agggccaagt tttccgcgag gtatccacaa cgccggcggc
     4741 cgcggtgtct cgcacacggc ttcgacggcg tttctggcgc gtttgcaggg ccatagacgg
     4801 ccgccagccc agcggcgagg gcaaccagcc cggtgagcgt cgcaaaggag atcctgatct
     4861 gactgatggg ctgcctgtat cgagtggtga ttttgtgccg agctgccggt cggggagctg
     4921 ttggctggct ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa
     4981 taacacattg cggacgtttt taatgtactg gggtggatgc agtgggccc
//
