LOCUS       sequence-245776-        6956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor vector for targeting a 2A peptide followed by blue fluorescent
            reporter to the human SCGB3A2 locus at the end of the endogenous 
            coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6956)
  AUTHORS   McCauley KB, Alysandratos KD, Jacob A, Hawkins F, Caballero IS, 
            Vedaie M, Yang W, Slovik KJ, Morley M, Carraro G, Kook S, Guttentag 
            SH, Stripp BR, Morrisey EE, Kotton DN
  TITLE     Single-Cell Transcriptomic Profiling of Pluripotent Stem 
            Cell-Derived SCGB3A2+ Airway Epithelium.
  JOURNAL   Stem Cell Reports. 2018 May 8;10(5):1579-1595. doi: 
            10.1016/j.stemcr.2018.03.013. Epub 2018 Apr 12.
  PUBMED    29657097
REFERENCE   2  (bases 1 to 6956)
  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"; doi: 
            "10.1016/j.stemcr.2018.03.013"; journalName: "Stem Cell Reports"; 
            date: "2018-05-8- 8"; volume: "10"; issue: "5"; pages: "1579-1595"
COMMENT     Sequence Label: pSCGB3A2_HL-P2A-TagBFP-PGK-NeoR-SCGB3A2_HR
FEATURES             Location/Qualifiers
     source          1..6956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..72
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     64..81
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     65..81
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     88..107
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        88..106
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     131..147
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     132..148
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             878..934
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             935..1636
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    1671..1704
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1765..2262
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1800..1823)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2195..2214
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2302..3105
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2365..2384)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2975..2994
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3137..3154)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3143..3367
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3440..3473
                     /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(3480..3496)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3480..3496)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(4233..4251)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4272..4288)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4272..4288)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4285..4307)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4296..4312
                     /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(4320..4326,4327..4344,4345..4350))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4365..4386
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4503..4520)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4674..5262)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4754..4773)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5433..6224,6225..6293))
                     /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     6056..6075
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6294..6398)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6424..6879)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6561..6582)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6773..6792
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
       61 cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga attgggtacc
      121 gggccccccc tcgaggtcga cggtatcgat aagcttgata tcgaattccg gctagcgtaa
      181 gatgagtgga tctggaatgg agctattgat ggacacaggg aaagaagatg aggtcatagt
      241 ctgggcccag atgtgcctgt ctcacctggt ttctcttttt cctgcagcta ctgccttcct
      301 catcaacaaa gtgccccttc ctgttgacaa gttggcacct ttacctctgg acaacattct
      361 tccctttatg gatccattaa agcttcttct gaaaactctg ggcatttctg ttgagcacct
      421 tgtggagggg ctaaggaagt gtgtaaatga gctgggacca gaggcttctg aagctgtgaa
      481 gaaactgctg gtaaccacag cttgggaggc taatctgcca aaggggaggc atactcaccc
      541 tgaatgtcta gctcctcttc ttgtcccttg taaatgtgca tttccacttt actgattata
      601 ttcataggaa gtttgcacat cctggaatct caggaaaaat acaatttcta catttctggg
      661 aagggttttt gaatgatagt gacatgtgtt atagttgtga caataccatt taccaccttc
      721 gtgattttgg atgagacgta atcactctga gtttttgttt ccccatcagt aaaatagtgg
      781 ccaaacagga cactggaagt gaatatgttt tgaaacatgc gaattacatg taagctgctc
      841 taaattttgt tgtccttcta catttcagga ggcgctagcc actaacttct ccctgttgaa
      901 acaagcaggg gatgtcgaag agaatcccgg gccaatgagc gagctgatta aggagaacat
      961 gcacatgaag ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga
     1021 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg
     1081 ccctctcccc ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt
     1141 catcaaccac acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac
     1201 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
     1261 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa
     1321 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc
     1381 cgctgacggc ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca
     1441 tctgatcgca aacatcaaga ccacatatag atccaagaaa cccgctaaga acctcaagat
     1501 gcctggcgtc tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac
     1561 ctacgtcgag cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg
     1621 gcacaagctt aattaaataa gaattccgat catattcaat aacccttaat ataacttcgt
     1681 ataatgtatg ctatacgaag ttattaggtc tgaagaggag tttacgtcca gccaagctta
     1741 ggatctcgac ctcgaaattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     1801 catgcgcttt agcagccccg ctggcacttg gcgctacaca agtggcctct ggcctcgcac
     1861 acattccaca tccaccggta gcgccaaccg gctccgttct ttggtggccc cttcgcgcca
     1921 ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg
     1981 acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag caccgctgag
     2041 caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc cttcgctttc
     2101 tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc
     2161 ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg
     2221 tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcat ccatctagat
     2281 ctcgagcagc tgaagcttac catgggatca gccattgaac aagatggatt gcacgcaggt
     2341 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2401 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2461 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     2521 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2581 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2641 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     2701 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     2761 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     2821 ttcgccaggc tcaaggcgcg catgcccgac ggcgatgatc tcgtcgtgac ccatggcgat
     2881 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     2941 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3001 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3061 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagggga tcaattctct
     3121 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3181 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3241 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3301 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3361 tctatggctt ctgaggcgga aaaaaccagc tggggctcga ctagagcttg cggaaccctt
     3421 catcagtcag gtacataata taacttcgta taatgtatgc tatacgaagt tattaggtgg
     3481 atccactagt tctagagcgg ccgccaccgc ggtggagctc cagcttttgt ttggtgtgac
     3541 atcaagataa agagcggagg tggatgggga tggaagatga tgctcctatc ctccctgcct
     3601 gaaacctgtt ctaccaatta tagatcaaat gccctaaaat gtagtgaccc gtgaaaagga
     3661 caaataaagc aatgaataca ttttcagtcg tcctattttt atttctgggg actctattca
     3721 gaaggacgta tccagatcag ttttcccttg gttcataaac tcgagagagc ctgtttttat
     3781 aaagaaaacc tggaatctta agtaagacat aatcattcct tctcctttcc ttatctctaa
     3841 tttaaacatc tctatttttc ccaaatgttt tatctcacgg tgttttcaga cccttcactt
     3901 tcctatcccc catcctaaat aaatttttac atttaaaaaa tgaaaaaata attttcaaaa
     3961 tattgtctga taagactagc atcttactaa cttcattggt ttggcttctg tttcactaga
     4021 atgctagttc ttaagtttgt agttaagcta tggccactca atagctttca ccaatccttc
     4081 caccaagtta ggcctttact acatcatcct taggcaaatg ggtgatgaac tatcttttct
     4141 ttcctttact ctgtcatcta cagtgatgac agaattatct ttccaaggaa ttattctggt
     4201 aatatcatga cggcctgtag agcagatttc tccctttagt gagggttaat ttcgagcttg
     4261 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4321 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4381 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4441 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4501 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4561 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4621 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4681 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4741 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4801 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4861 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4921 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4981 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5041 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5101 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5161 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5221 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5281 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5341 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5401 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5461 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5521 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5581 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5641 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5701 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5761 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5821 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5881 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5941 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6001 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6061 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6121 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6181 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6241 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6301 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6361 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6421 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     6481 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6541 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6601 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     6661 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6721 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6781 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     6841 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     6901 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcg
//
