LOCUS       sequence-465740-        4953 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 POC1343, POC1344, POC1345 and POC1346; respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4953)
  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 4953)
  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: POC1355
FEATURES             Location/Qualifiers
     source          1..4953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    87..108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(123..128,129..146,147..153)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    161..177
                     /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     166..188
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     185..201
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     185..201
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             197..520
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    214..270
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(271..288)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(271..287)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(280..302)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(496..515)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    659..683
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(839..1987)
                     /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(2323..2911)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2403..2422)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3082..3873,3874..3942))
                     /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     3705..3724
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3943..4047)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4134..4765
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4857..4881
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 cactctgtga cttggtctca tgcctgagac cgcagctggc acgacaggtt tcccgactgg
       61 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      121 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      181 cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca ggtcgactct
      241 agaggatccc cgggtaccga gctcgaattc actggccgtc gttttacaac gtcgtgactg
      301 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
      361 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
      421 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
      481 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgaccccc
      541 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
      601 agctgtgacg gtctcacaga tgagaccaag tcacgaagtg gtttaaacta tcagtgtttg
      661 acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat aatcggatat
      721 ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg
      781 gttccccaga tcaggcgctg gctgctgaac ccccagccgg aactgacccc acaaggccct
      841 agcgtttgca atgcaccagg tcatcattga cccaggcgtg ttccaccagg ccgctgcctc
      901 gcaactcttc gcaggcttcg ccgacctgct cgcgccactt cttcacgcgg gtggaatccg
      961 atccgcacat gaggcggaag gtttccagct tgagcgggta cggctcccgg tgcgagctga
     1021 aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg gtacttctcc catatgaatt
     1081 tcgtgtagtg gtcgccagca aacagcacga cgatttcctc gtcgatcagg acctggcaac
     1141 gggacgtttt cttgccacgg tccaggacgc ggaagcggtg cagcagcgac accgattcca
     1201 ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc ctgtaggcgc gacaggcatt
     1261 cctcggcctt cgtgtaatac cggccattga tcgaccagcc caggtcctgg caaagctcgt
     1321 agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt cgcgtactcc aacacctgct
     1381 gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc ggtgtaggtg atcttcacgt
     1441 ccttgttgac gtggaaaatg accttgtttt gcagcgcctc gcgcgggatt ttcttgttgc
     1501 gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat cgctcgcatc gtgtccggcc
     1561 acggcgcaat atcgaacaag gaaagctgca tttccttgat ctgctgcttc gtgtgtttca
     1621 gcaacgcggc ctgcttggcc tcgctgacct gttttgccag gtcctcgccg gcggtttttc
     1681 gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat cgacttcgcc aaacctgccg
     1741 cctcctgttc aagacgacgc gaacgctcca cggcggccga tggcgcgggc agggcagggg
     1801 gagccagttg cacgctgtcg cgctcgatct tggccgtagc ttgctggacc atcgagccga
     1861 cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg gcttgcgatg gtttcggcat
     1921 cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta tgccttccgg tcaaacgtcc
     1981 gattcattca ccctccttgc gggattgccc cgactcacgc cggggcaatg tgcccttatt
     2041 cctgatttga cccgcctggt gccttggtgt ccagataatc caccttatcg gcaatgaagt
     2101 cggtcccgta gaccgtctgg ccgtccttct cgtacttggt attccgaatc ttgccctgca
     2161 cgaataccag cgaccccttg cccaaatact tgccgtgggc ctcggcctga gagccaaaac
     2221 acttgatgcg gaagaagtcg gtgcgctcct gcttgtcgcc ggcatcgttg cgccacatct
     2281 aggatctgcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2341 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2401 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2461 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2521 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2581 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2641 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2701 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2761 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2821 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2881 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2941 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3001 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3061 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3121 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3181 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagaaccac gctcaccggc
     3241 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3301 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3361 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3421 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3481 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     3541 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3601 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3661 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3721 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3781 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3841 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3901 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3961 ttattgaagc atttatcaag gttattgtct catgagcgga tacatatttg aatgtattta
     4021 gaaaaataaa caaatagggg ttccgcgcac gaattggcca gcgctgccat ttttggggtg
     4081 aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggaggcccgc gttagcgggc
     4141 cgggagggtt cgagaagggg gggcaccccc cttcggcgtg cgcggtcacg cgcacagggc
     4201 gcagccctgg ttaaaaacaa ggtttataaa tattggttta aaagcaggtt aaaagacagg
     4261 ttagcggtgg ccgaaaaacg ggcggaaacc cttgcaaatg ctggattttc tgcctgtgga
     4321 cagcccctca aatgtcaata ggtgcgcccc tcatctgtca gcactctgcc cctcaagtgt
     4381 caaggatcgc gcccctcatc tgtcagtagt cgcgcccctc aagtgtcaat accgcagggc
     4441 acttatcccc aggcttgtcc acatcatctg tgggaaactc gcgtaaaatc aggcgttttc
     4501 gccgatttgc gaggctggcc agctccacgt cgccggccga aatcgagcct gcccctcatc
     4561 tgtcaacgcc gcgccgggtg agtcggcccc tcaagtgtca acgtccgccc ctcatctgtc
     4621 agtgagggcc aagttttccg cgaggtatcc acaacgccgg cggccgcggt gtctcgcaca
     4681 cggcttcgac ggcgtttctg gcgcgtttgc agggccatag acggccgcca gcccagcggc
     4741 gagggcaacc agcccggtga gcgtcgcaaa ggagatcctg atctgactga tgggctgcct
     4801 gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct ggctggtggc
     4861 aggatatatt gtggtgtaaa caaattgacg cttagacaac ttaataacac attgcggacg
     4921 tttttaatgt actggggtgg atgcagtggg ccc
//
