LOCUS       sequence-394155-        7369 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockdown of Collagen Type 1 alpha 1. Construct has inverted repeats
            to be used in Sleeping Beauty transposon system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7369)
  AUTHORS   Comba A, Faisal SM, Dunn PJ, Argento AE, Hollon TC, Al-Holou WN, 
            Varela ML, Zamler DB, Quass GL, Apostolides PF, Abel C 2nd, Brown 
            CE, Kish PE, Kahana A, Kleer CG, Motsch S, Castro MG, Lowenstein PR
  TITLE     Spatiotemporal analysis of glioma heterogeneity reveals COL1A1 as an
            actionable target to disrupt tumor progression.
  JOURNAL   Nat Commun. 2022 Jun 24;13(1):3606. doi: 10.1038/s41467-022-31340-1.
  PUBMED    35750880
REFERENCE   2  (bases 1 to 7369)
  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: "Nat 
            Commun."; date: "2022-06-24"; volume: "13(1):3606. doi"; pages: " 
            10.1038/s41467-022-31340-1"
COMMENT     Sequence Label: pT2/sh-Col1a1/GFP4_Seq1.2
FEATURES             Location/Qualifiers
     source          1..7369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(94..113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     304..325
                     /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"
     LTR             1034..1550
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    1614..1955
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             2022..2438
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     2368..2390
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     2406..2422
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           2478..2599
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2626..2640
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2667..2793
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(2826..2846)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2831..3160
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3011..3146
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3167..3169,3170..3172,3173..3886)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3212..3233)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3473..3492)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3820..3841
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3959..4473
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4635..4651
                     /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(4659..4665,4666..4683,4684..4689))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4704..4725
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5184..5204)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5184..5202)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5223..5239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5223..5239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5247..5263
                     /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(5271..5277,5278..5295,5296..5301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5316..5337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5454..5471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5625..6213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5705..5724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6384..7175,7176..7244))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7007..7026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7245..7349)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg agtggtggtt acgcgcagcg
       61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      361 atttataagg gattttgccg atttcggcct attggttaaa 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 aattggagct
      661 cggatcccta tacagttgaa gtcggaagtt tacatacact taagttggag tcattaaaac
      721 tcgtttttca actactccac aaatttcttg ttaacaaaca atagttttgg caagtcagtt
      781 aggacatcta ctttgtgcat gacacaagtc atttttccaa caattgttta cagacagatt
      841 atttcactta taattcactg tatcacaatt ccagtgggtc agaagtttac atacactaag
      901 ttgactgtgc ctttaaacag cttggaaaat tccagaaaat gatgtcatgg ctttagaagc
      961 ttgatctgaa gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc
     1021 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
     1081 cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg
     1141 aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     1201 ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag
     1261 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
     1321 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
     1381 aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta
     1441 cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt
     1501 gggagggtct cctcagattg attgactgcc cacctcgggg gtctttcatt tggaggttcc
     1561 accgagattt ggagacccct gcccagggac caccgacccc cccgccggga ggtaagctgg
     1621 ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt
     1681 tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
     1741 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
     1801 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
     1861 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     1921 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
     1981 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
     2041 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     2101 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     2161 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     2221 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     2281 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     2341 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     2401 cctcccttta tccagccctc actccttctc taggcgccgg aattagatct ctcgactagg
     2461 gataacaggg taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat
     2521 cttggaaaca cttgctggga ttacttcttc aggttaaccc aacagaaggc tcgagaaggt
     2581 atattgctgt tgacagtgag cggcaacagt cgcttcacct acatagtgaa gccacagatg
     2641 tatgtaggtg aagcgactgt tgctgcctac tgcctcggaa ttcaaggggc tactttagga
     2701 gcaattatct tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa
     2761 gctgaattaa aatggtataa attaaatcac ttttttcaat tcgccagcac agtggtcgac
     2821 cctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2881 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2941 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3001 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3061 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3121 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcatgg tgagcaaggg
     3181 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     3241 ccacaagttc agcgtgtctg gcgagggcga gggcgatgcc acctacggca agctgaccct
     3301 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     3361 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     3421 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     3481 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     3541 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     3601 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggcgaa
     3661 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     3721 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     3781 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3841 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaatgaa ttaattaaga
     3901 attatcaagc ttatcgataa aataaaagat tttatttagt ctccagaaaa aggggggaat
     3961 gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg
     4021 gaaaatacat aactgagaat agagaagttc agatcaaggt taggaacaga gagacagcag
     4081 aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa
     4141 cagatggtcc ccagatgcgg tcccgccctc agcagtttct agagaaccat cagatgtttc
     4201 cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg
     4261 cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc
     4321 tcactcggcg cgccagtcct ccgatagact gcgtcgcccg ggtacccgtg tatccaataa
     4381 accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga
     4441 gtgattgact acccgtcagc gggggtcttt catgggtaac agtttcttga agttggagaa
     4501 caacattctg agggtaggag tcgaatatta agtaatcctg actcaattag ccactgtttt
     4561 gaatccacat actccaatac tcctgaaata gttcattatg gacagcgcag aaagagctgg
     4621 ggagaattgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4681 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4741 ctgcccgctt tccagtcggg aaacctgtcg tgccagatcc atggacttca ctagtgcgcg
     4801 cggccgctct agatggccag atctagcttg tggaaggcta ctcgaaatgt ttgacccaag
     4861 ttaaacaatt taaaggcaat gctaccaaat actaattgag tgtatgtaaa cttctgaccc
     4921 actgggaatg tgatgaaaga aataaaagct gaaatgaatc attctctcta ctattattct
     4981 gatatttcac attcttaaaa taaagtggtg atcctaactg acctaagaca gggaattttt
     5041 actaggatta aatgtcagga attgtgaaaa agtgagttta aatgtatttg gctaaggtgt
     5101 atgtaaactt ccgacttcaa ctgtataggg atcctctagc tagagtcgac ctcgcggggg
     5161 ggcccggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     5221 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5281 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5341 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5401 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5461 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5521 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5581 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5641 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5701 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5761 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaaccgtggc gctttctcat
     5821 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5881 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5941 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6001 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6061 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6121 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6181 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6241 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6301 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6361 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6421 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6481 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6541 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6601 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6661 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6721 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6781 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6841 aacttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6901 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6961 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7021 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7081 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7141 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7201 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7261 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7321 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgc
//
