LOCUS       sequence-472079-        4958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PaqCI Golden Gate assembly plasmid encoding the backbone for 
            Bxb1-mediated integration in the Rogi2 landing pad line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4958)
  AUTHORS   Peterman EL, Ploessl DS, Love KS, Sanabria V, Daniels RF, Johnstone 
            CP, Godavarti DR, Kabaria SR, Oakes CG, Pai AA, Galloway KE
  TITLE     High-resolution profiling reveals coupled transcriptional and 
            translational regulation of transgenes.
  JOURNAL   Nucleic Acids Research, Volume 53, Issue 11, 24 June 2025, gkaf528
  PUBMED    39651241
REFERENCE   2  (bases 1 to 4958)
  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 Research, Volume 53, Issue 11, 24 June 2025, gkaf528"
COMMENT     Sequence Label: pKG3560_pHarbor-Rogi2_backbone
FEATURES             Location/Qualifiers
     source          1..4958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2..19)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2..18)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        31..135
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(136..204,205..996)
                     /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     complement(354..373)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1167..1755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1656..1675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1838..3016
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2069..3007
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2964..2984
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3024..3033
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3036..3073
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(3082..3115)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3173..3297
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3330..3335,3336..3353,3354..3360)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3414..4094
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(3480..3499)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4414..4719
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4598..4617
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4756..4880)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(4896..4929)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
ORIGIN
        1 cactggccgt cgttttacaa ctataatgtg cgcggaaccc ctatttgttt atttttctaa
       61 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
      121 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
      181 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
      241 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
      301 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
      361 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
      421 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
      481 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
      541 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
      601 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
      661 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
      721 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
      781 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
      841 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
      901 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
      961 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     1021 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     1081 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     1141 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     1201 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1261 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     1321 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1381 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1441 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1501 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1561 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1621 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1681 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1741 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1801 ccttttgctc acatgttctt tcctgcgtta tcgaattggc tccggtgccc gtcagtgggc
     1861 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
     1921 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     1981 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2041 tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg
     2101 cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt
     2161 gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta
     2221 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
     2281 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
     2341 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
     2401 ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
     2461 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
     2521 ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc
     2581 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
     2641 gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc
     2701 gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga
     2761 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac
     2821 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     2881 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     2941 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     3001 atttcaggtg tcgtgaggaa ttcgccacca tggccggctt gtcgacgacg gcggtctccg
     3061 tcgtcaggat catccggatc cataacttcg tatagcatac attatacgaa gttatcatga
     3121 tattcggcaa gcaggcatcg tactcatagc aggtgccact agtggagttc cgacaagttt
     3181 gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
     3241 tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt cactatggcg
     3301 gattcgcggc cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg
     3361 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
     3421 aaaaaatcac tggatatacc acagttgata tatcccaatg gcatcgtaaa gaacattttg
     3481 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     3541 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     3601 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
     3661 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     3721 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     3781 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     3841 ttttcgtttc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     3901 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     3961 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     4021 gaatgcttaa tgaattacaa cagtactgcg atgagtggca ggcggggcgt aatctagagg
     4081 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     4141 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     4201 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     4261 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     4321 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     4381 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     4441 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     4501 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt cccccgtgaa
     4561 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     4621 agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     4681 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     4741 agcctgcagg tcgaccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt
     4801 tttttatgca aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca
     4861 gctttcttgt acaaagtggt cacctgctat gcgctataac ttcgtatagg atactttata
     4921 cgaagttatc accagagacc gtccggggtc tctgcgct
//
