LOCUS       sequence-394842-        5224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination plasmid for Matreyek Bxb1(GT) landing pad in dual 
            landing pad cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5224)
  AUTHORS   Roelle SM, Kamath ND, Matreyek KA
  TITLE     Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase 
            Sites for the Functional Characterization of Protein Variants.
  JOURNAL   ACS Synth Biol. 2023 Nov 3. doi: 10.1021/acssynbio.3c00355.
  PUBMED    37922210
REFERENCE   2  (bases 1 to 5224)
  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: "ACS Synth 
            Biol. 2023 Nov 3. doi: 10.1021/acssynbio.3c00355."
COMMENT     Sequence Label: 
            J135F_BattB_Vif(T27X4)-IRES-SpyTag-sfmCherry2(11)-PuroR
FEATURES             Location/Qualifiers
     source          1..5224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     432..451
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(670..774)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(800..1255)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(937..958)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1149..1168
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1382..1404
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1396..1413
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1397..1413
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1420..1439
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1420..1438
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1512..1549
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      1574..1583
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(2280..2854,2855..2857,2858..2866)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2447..2464)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2674..2693
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2870..2911
                     /codon_start=1
                     /product="improved SpyTag variant that shows rapid reaction
                     with SpyCatcher002 (Keeble et al., 2017)"
                     /label=SpyTag002
                     /translation="VPTIVMVDAYKRYK"
     primer_bind     3492..3512
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3703..3824
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3740..3759)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3794..3813
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(3833..3851)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3872..3888)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3872..3888)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3885..3907)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3896..3912
                     /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(3920..3926,3927..3944,3945..3950))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3965..3986
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4103..4120)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4274..4862)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4354..4373)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5033..5224,1..600,601..669))
                     /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"
ORIGIN
        1 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
       61 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      121 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      181 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      241 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
      301 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
      361 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      421 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      481 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
      541 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
      601 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
      661 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
      721 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
      781 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
      841 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
      901 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
      961 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     1021 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     1081 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     1141 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     1201 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     1261 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     1321 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     1381 tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc
     1441 gaattgggta ccgggccccc cctcgaggtc gacgatgtag gtcacggcat tccggactca
     1501 gcgctcgagc cggcttgtcg acgacggcgg tctccgtcgt caggatcatc cgcagattat
     1561 accgcaacta cacgccacca tggaaaacag atggcaggtg atgattgtgt ggcaagtaga
     1621 caggatgagg attagaacat ggaaaagttt agtaaaatag taatgatagg tttcagggaa
     1681 agctagggga tggttttata gacatcacta tgaaagccct catccaagaa taagttcaga
     1741 agtacacatc ccactagggg atgctagatt ggtaataaca acatattggg gtctgcatac
     1801 aggagaaaga gactggcatt tgggtcaggg agtctccata gaatggagga aaaagagata
     1861 tagcacacaa gtagaccctg aactagcaga ccaactaatt catctgtatt actttgactg
     1921 tttttcagac tctgctataa gaaaggcctt attaggacac atagttagcc ctaggtgtga
     1981 atatcaagca ggacataaca aggtaggatc tctacaatac ttggcactag cagcattaat
     2041 aacaccaaaa aagataaagc cacctttgcc tagtgttacg aaactgacag aggatagatg
     2101 gaacaagccc cagaagacca agggccacag agggagccac acaatgaatg gacactagta
     2161 acttaagaat tcaccggtct gacctccttc tccctctctt caggtctgca gtcgacggta
     2221 ccgcgggccc gggatccacc ggatctagat aactgatcac tgtggaatca gagagactgc
     2281 ccctctccct ctcccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     2341 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     2401 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     2461 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     2521 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     2581 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     2641 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     2701 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     2761 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccatggg
     2821 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatgg tgcctactat
     2881 cgtgatggtg gacgcctaca agcgttacaa gggcggatat acaatcgtcg agcagtatga
     2941 acgggcggag gctaggcaca gcaccggtgg atcaggaggc ggttcgtcag gttctggcac
     3001 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     3061 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     3121 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     3181 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     3241 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3301 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3361 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     3421 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     3481 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     3541 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagtaatagg gatcctgatc
     3601 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     3661 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     3721 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3781 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaggagat cccctttagt
     3841 gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3901 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3961 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4021 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4081 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcactcggtc gttcggctgc
     4141 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4201 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4261 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4321 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4381 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4441 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4501 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4561 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4621 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4681 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4741 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4801 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4861 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4921 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4981 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5041 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5101 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5161 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5221 ccgg
//
