LOCUS       sequence-390547-        4830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To create ""long homology arm"" (~1000bp) knock-in donor vectors for
            split-intein Gal4[1-20]N-int.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4830)
  AUTHORS   Ewen-Campen B, Luan H, Xu J, Singh R, Joshi N, Thakkar T, Berger B, 
            White BH, Perrimon N
  TITLE     split-intein Gal4 provides intersectional genetic labeling that is 
            repressible by Gal80.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Jun 13;120(24):e2304730120. doi: 
            10.1073/pnas.2304730120. Epub 2023 Jun 5.
  PUBMED    37276389
REFERENCE   2  (bases 1 to 4830)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-06-13"; volume: "120(24):e2304730120. 
            doi"; pages: " 10.1073/pnas.2304730120. Epub 2023 Jun 5"
COMMENT     Sequence Label: pHD-T2A-split-intein-Gal4[1-20]-N-int
FEATURES             Location/Qualifiers
     source          1..4830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      6..462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(93..112)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     303..324
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             740..793
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    1378..1411
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1637..1646
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1643..2320
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(1773..1791)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1823..1843)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2041..2060
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2235..2258
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2441..2562
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2478..2497)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2532..2551
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2592..2625
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2644..2664)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2644..2662)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2683..2699)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2683..2699)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2696..2718)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2707..2723
                     /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(2731..2737,2738..2755,2756..2761))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2776..2797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2914..2931)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3085..3673)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3165..3184)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3844..4635,4636..4704))
                     /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     4467..4486
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4705..4809)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
       61 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
      121 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
      181 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
      241 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
      301 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
      361 tttataaggg attttgccga tttcggccta ttggttaaaa aaatgagctg atttaacaaa
      421 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
      661 aagcttcacg acctgaggcg cgccgggcca gtacccaaaa agcggggggt ccggtggaag
      721 cggaggtagc gcggccgccg agggccgcgg cagcctgctg acctgcggcg atgtggagga
      781 gaaccccggg cccgctagca tgaagctact gtcttctatc gaacaagcat gcgatatttg
      841 ccgacttaaa aagctcaagt gcctggatct gaagacccag gtgcagaccc cccagggcat
      901 gaaggagatc agcaacatcc aggtgggcga tctggtgctg agcaacaccg gctacaacga
      961 ggtgctgaac gtgttcccca agagcaagaa gaagagctac aagatcaccc tggaggatgg
     1021 caaggagatc atctgcagcg aggagcacct gttccccacc cagaccggcg agatgaacat
     1081 cagcggcggc ctgaaggagg gcatgtgcct gtacgtgaag gagtaacctc tagagtcgac
     1141 taaagccaaa tagaaattat tcagttctgg cttaagtttt taaaagtgat attatttatt
     1201 tggttgtaac caaccaaaag aatgtaaata actaatacat aattatgtta gttttaagtt
     1261 agcaacaaat tgattttagc tatattagct acttggttaa taaatagaat atatttattt
     1321 aaagataatt cgtttttatt gtcagggagt gagtttgctt aaaaactcgt ttggatcata
     1381 acttcgtata atgtatgcta tacgaagtta tcgtacggga tctaattcaa ttagagacta
     1441 attcaattag agctaattca attaggatcc aagcttatcg atttcgaacc ctcgaccgcc
     1501 ggagtataaa tagaggcgct tcgtctacgg agcgacaatt caattcaaac aagcaaagtg
     1561 aacacgtcgc taagcgaaag ctaagcaaat aaacaagcgc agctgaacaa gctaaacaat
     1621 cggctcgaag ccggtcgcca ccatggcctc ctccgaggac gtcatcaagg agttcatgcg
     1681 cttcaaggtg cgcatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     1741 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc
     1801 cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca aggtgtacgt
     1861 gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg
     1921 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     1981 ccaggacggc tccttcatct acaaggtgaa gttcatcggc gtgaacttcc cctccgacgg
     2041 ccccgtaatg cagaagaaga ctatgggctg ggaggcgtcc accgagcgcc tgtacccccg
     2101 cgacggcgtg ctgaagggcg agatccacaa ggccctgaag ctgaaggacg gcggccacta
     2161 cctggtggag ttcaagtcca tctacatggc caagaagccc gtgcagctgc ccggctacta
     2221 ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacacca tcgtggagca
     2281 gtacgagcgc gccgagggcc gccaccacct gttcctgtag gggccgcgac tctagatcat
     2341 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2401 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2461 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2521 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaccgtctg taagcagcga
     2581 aggcccatac gataacttcg tataatgtat gctatacgaa gttatgagct ccagcttttg
     2641 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     2701 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2761 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     2821 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2881 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2941 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3001 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3061 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3121 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3181 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3241 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3301 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3361 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3421 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3481 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3541 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3601 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3661 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3721 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3781 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3841 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3901 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3961 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4021 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4081 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4141 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4201 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4261 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4321 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4381 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4441 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4501 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4561 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4621 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4681 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4741 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4801 ggttccgcgc acatttcccc gaaaagtgcc
//
